Subcutaneous delivery of polymer conjugates of therapeutic agents

ABSTRACT

The present disclosure provides polymer conjugates comprising a polymer and an agent, the agent linked to the polymer via a linking group containing a cleavable moiety.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.14/355,515, filed May 1, 2014 (currently pending). U.S. application Ser.No. 14/355,515 is a 371 of International Application No. PCT/US12/63088,filed Nov. 1, 2012 (currently expired), PCT/US12/63088 is a continuationin part of U.S. application Ser. No. 13/524,994 filed Jun. 15, 2012, nowU.S. Pat. No. 8,383,093, issued on Feb. 26, 2013, which claims thebenefit of U.S. Provisional Application No. 61/554,336, filed Nov. 1,2011 (currently expired).

FIELD OF THE DISCLOSURE

The present disclosure is related generally to polymer conjugates. Thepresent disclosure relates more specifically to polymer conjugatescomprising a water soluble polymer and an agent, the agent linked to thewater soluble polymer by a releasable linker, the releasable linkercomprising a cleavable moiety which is cleavable in a subject to releasethe agent after administration of the conjugate to a subject. Methods ofusing such conjugates for treatment and methods for the preparation ofsuch conjugates are also provided.

BACKGROUND

Development of drug conjugates with water-soluble polymers can enhancethe properties of the drugs, including water-solubility,pharmacokinetics, metabolism, bio-distribution, and bioactivity. Anumber of polymer-protein conjugates having stable linkages have beenapproved by FDA and are currently valuable medicines (Bentley, M. D. etal., Poly(ethylene) Glycol Conjugates of Biopharmaceuticals in DrugDelivery, in Knablein, J. (ed.), Modem Biopharmaceuticals, Wiley-VCHVerlag GbH, Volume 4, 2005, Chapter 2, pp. 1393-1418). Conjugation ofwater-soluble polymers including poly(ethylene glycol), poly(glutamate),and poly(hydroxypropylmethacrylate) with small molecule oncolytics hasled to several products in clinical trials, but as yet, no marketeddrugs (Mero, A., PEG: a useful technology in anticancer therapy, inVeronese, F. M. (ed.), PEGylated Protein Drugs: Basic Science andClinical Application, Birkhauser Verlag, Basel, 2009, pp. 273-281).Unlike the case of protein conjugates, it is frequently useful toformulate small-molecule conjugates with releasable linkages. Thesepolymer conjugates are known to significantly extend the half-lives ofthe attached small molecules. When the oncolytic drug, irinotecan, wasattached to a multi-arm polyethylene glycol polymer, and injectedintravenously to mice the plasma half-life of its active metaboliteSN-38 was increased from 2 hours to 17 days (Eldon, M. A. et al.,Anti-tumor activity and pharmacokinetics of NKTR-102,PEGylated-irinotecan conjugate, in irinotecan-resistant tumors implantedin mice, Poster number: P-0722, presented at the 14th European CancerConference (ECCO 14), 23-27 Sep. 2007, Barcelona, Spain).

The advantage of polymer conjugates of small molecule drugs derives fromthe typically short in vivo half-life of the drug. The short half-livesof these drugs require frequent dosing of several times daily whichresults in “pulses” of high concentration of the drug, followed bylonger periods where the drug concentration in the blood stream is belowthe amount required for therapeutic efficacy. For example, in somecases, such as Parkinson's disease (PD), pulsatile stimulation ofstriatal dopamine receptors with short-acting dopamine agonists orlevo-dopa may actually accelerate molecular and physiological changesthat lead to degeneration of dopaminergic neurons in the central nervoussystem (CNS), thus promoting motor fluctuations (dyskinesias) that canbe disabling. Physiological levels that are maintained at a steady statewithout phasic peak and trough levels have been shown to eliminate theseside effects in both animals and humans. Low solubility of some of thesecompounds, combined with limited oral bioavailabity, further complicatestheir clinical use. These problems may be solved by preparation of asoluble polymer conjugate.

The art is lacking a composition that is administered by thesubcutaneous route and is able to provide sustained, controllabledelivery of a drug over a period of days to weeks. The presentdisclosure provides polymer conjugates comprising a water solublepolymer and an agent, the agent linked to the water soluble polymer by areleasable linker, the releasable linker comprising a cleavable moietywhich is cleavable in a subject to release the agent afteradministration of the conjugate to a subject. The present disclosureprovides such conjugates. As shown herein, the subcutaneous injection ofsuch polymer conjugates provides sustained delivery of the agent attherapeutically effective levels of a drug over a time period of days toweeks.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows an HPLC chromatogram of rotigotine 2-azidoacetate beforereversed phase chromatography purification

FIG. 1B shows an HPLC chromatogram of rotigotine 2-azidoacetate afterreversed phase chromatography purification

FIG. 2 shows the pharmacokinetic profile of rotigotine after intravenousdosing of POZ rotigotine in male Sprauge-Dawley rats.

FIG. 3 shows the pharmacokinetic profile of rotigotine aftersubcutaneous dosing of POZ rotigotine in male Sprauge-Dawley rats.

FIG. 4 shows the pharmacokinetic profile of rotigotine aftersubcutaneous dosing of POZ-rotigotine in female Cynomolgus monkeys.

SUMMARY OF THE DISCLOSURE

In a first aspect, the present disclosure provides a polymer conjugatecomprising a water-soluble polymer and an agent, the agent linked to thepolymer by a releasable linker. In certain embodiments of this aspect,the agent is a diagnostic agent or a therapeutic agent, such as, but notlimited to, an organic small molecule.

In a second aspect, the present disclosure provides a polymer conjugatecomprising a water-soluble polymer and an agent useful in the treatmentof Parkinson's Disease (PD) or other diseases or conditions related todopamine insufficiency in the peripheral or central nervous system inwhich the agent is linked to the polymer by a releasable linker.

In a third aspect, the present disclosure provides a polymer conjugatecomprising a water-soluble polymer and an agent useful in the treatmentof a disorder characterized by excessive GABA re-uptake or GABAre-uptake or an anxiety disorder, social anxiety disorder, panicdisorder, neuropathic pain, chronic pain, muscle tremors, muscle spasms,seizures, convulsions and/or epilepsy in which said inhibitor is linkedto the polymer by a releasable linker.

In a fourth aspect, the present disclosure provides a polymer conjugatecomprising a water-soluble polymer and a dopamine agonist in which thedopamine agonist is linked to the polymer by a releasable linker or awater-soluble polymer and a GABA re-uptake inhibitor in which the GABAre-uptake inhibitor is linked to the polymer by a releasable linker.

In a fifth aspect, the present disclosure provides a polymer conjugatecomprising a water-soluble polymer and rotigotine, the rotigotine linkedto the polymer by a releasable linker, a polymer conjugate comprising awater-soluble polymer and ropinirole, the ropinirole linked to thepolymer by a releasable linker and a polymer conjugate comprising awater-soluble polymer and tiagabine, the tiagabine linked to the polymerby a releasable linker. In one embodiment of the foregoing, the .watersoluble polymer is polyoxazoline, dextran, dextran modified by oxidationor polyethylene glycol.

In a sixth aspect, the present disclosure provides a poly(oxazoline)(POZ) conjugate comprising a POZ polymer and an agent, the agent linkedto the POZ polymer by a releasable linker. In certain embodiments ofthis aspect, the agent is a diagnostic agent or a therapeutic agent,such as, but not limited to, an organic small molecule.

In a seventh aspect, the present disclosure provides a POZ polymerconjugate comprising a POZ polymer and an agent useful in the treatmentof PD or other diseases or conditions related to dopamine insufficiencyin the peripheral or central nervous system, the agent linked to thepolymer by a releasable linker.

In an eighth aspect, the present disclosure provides a POZ polymerconjugate comprising a POZ polymer and an agent useful in the treatmentof a disorder characterized by excessive GABA re-uptake or GABAre-uptake or an anxiety disorder, social anxiety disorder, panicdisorder, neuropathic pain, chronic pain, muscle tremors, muscle spasms,seizures, convulsions and/or epilepsy in which said inhibitor is linkedto the polymer by a releasable linker.

In ninth aspect, the present disclosure provides a POZ polymer conjugatecomprising a POZ polymer and a dopamine agonist, the dopamine agonistlinked to the POZ polymer by a releasable linker or and a POZ polymerand a GABA re-uptake, the GABA re-uptake inhibitor is linked to the POZpolymer by a releasable linker.

In a tenth aspect, the present disclosure provides a POZ polymerconjugate comprising a POZ polymer and rotigotine, the rotigotine linkedto the POZ polymer by a releasable linker, a POZ polymer conjugatecomprising a POZ polymer and ropinirole, the ropinirole linked to thePOZ polymer by a releasable linker and a POZ polymer conjugatecomprising a POZ polymer and tiagabine, the tiagabine linked to the POZpolymer by a releasable linker.

In any of the first through fifth aspects, the water-soluble polymer maybe a water soluble polymer known in the art. Exemplary water solublepolymers suitable for use with the present disclosure include, but arenot limited to, the following water-soluble polymers: POZ,poly(5,6-dihydro-4h-1,3-oxazine), dextran, dextran modified byoxidation, polyethylene glycol (PEG), poly(hydroxypropylmethacrylate),polyglutamic acid, polylactic-polyglutamic acid mixture, polysialicacid, polycaprolactone, polyvinylpyrrolidone, poly(sialic acid),polyglycosaminoglycan, polyglycerol,poly(acryloyloxyethylphosphorylcholine), and methacrylate-basedcopolymer with synthetic forms of phosphorylcholine. Combinations of theforegoing are also included. In a particular embodiment of the firstthrough fifth aspects, the water-soluble polymer is POZ, PEG, dextran ordextran modified by oxidation. In another particular embodiment of thefirst through fifth aspects, the water-soluble polymer is POZ. Inanother embodiment, of the first through fifth aspects, thewater-soluble polymer is a copolymer of PEG and POZ.

In any of the first through tenth aspects, the releasable linkercontains a cleavable moiety, the cleavable moiety being optionallycontained in a larger chemical moiety (i.e, a linking group), allowingthe chemical linkage between the agent and the polymer to be cleaved. Incertain embodiments of this aspect, the cleavable moiety is an ester, acarbonate ester, a carboxylate ester, a carbamate, a disulfide, anacetal, a hemiacetal, a phosphate, a phosphonate or an amide. In aparticular embodiment, the cleavable moiety is an ester. Suitable esterfunctionalities include, but are not limited to, carboxylate ester andcarbonate esters.

In any of the foregoing aspects, exemplary agents useful in thetreatment of PD or other diseases or conditions related to dopamineinsufficiency in the peripheral or central nervous include, but are notlimited to, dopamine agonists, adenosine A_(2A) antagonist,anticholinergics, monamine oxidase-B inhibitors and catechol-O-methyltransferase (COMT) inhibitors. Exemplary dopamine agonists include, butare not limited to, rotigotine, pramipexole, quinagolide, fenoldopam,apomorphine, 5-OH-DPAT, ropinirole, pergolide, cabergoline, andbromocriptine. Exemplary anticholinergics include, but are not limitedto, trihexyphenidyl, biperidin and hyoscyamine. Exemplary monamineoxidase-B inhibitors include, but are not limited to, seligiline andrasagiline. Exemplary COMT inhibitors include, but are not limited to,tolcapone and entacapone. Exemplary A2a antagonists include, but are notlimited to, caffeine, theophylline, istradefylline, and preladenant.

In any of the foregoing aspects, exemplary GABA re-uptake inhibitorinclude, but are not limited to, tiagabine and nipecotic acid. In any ofthe third, fourth, eighth or ninth aspects, the GABA re-uptake inhibitoris tiagabine.

In any of the foregoing aspects, exemplary dopamine agonists include,but are not limited to, rotigotine, pramipexole, quinagolide,fenoldopam, apomorphine, 5-OH-DPAT, ropinirole, pergolide, cabergoline,and bromocriptine. In any of the second, fourth, seventh or ninthaspects, the dopamine agonist is rotigotine. In any of the second,fourth, seventh or ninth aspects, the dopamine agonist is (-)rotigotine.

In any of the first through tenth aspects, the agent may be a diagnosticagent or a therapeutic agent. In any of the first through tenth aspects,the therapeutic agent may be an organic small molecule.

In an eleventh aspect, the present disclosure provides a method oftreatment for a disease, the method comprising the steps ofadministering a conjugate of the first through tenth aspects to asubject.

In a twelfth aspect, the present disclosure provides a method oftreatment for a disease, the method comprising the step of administeringa conjugate of the first through tenth aspects to a subject, wherein thelevel of the agent in the bloodstream is controlled by the nature of theagent, the nature of the linking group, the nature of the polymer, thesize of the polymer, the method of delivery or a combination of theforegoing.

In a thirteenth aspect, the present disclosure provides a method oftreatment for PD or other diseases or conditions related to dopamineinsufficiency in the peripheral or central nervous system, the methodcomprising the step of administering a conjugate of the first-second,fourth-seventh or ninth-tenth aspects to a subject.

In an fourteenth aspect, the present disclosure provides a method oftreatment for PD or other diseases or conditions related to dopamineinsufficiency in the peripheral or central nervous system, the methodcomprising the step of administering a conjugate of the first-second,fourth-seventh or ninth-tenth aspects to a subject, wherein the levelsof the agents in the bloodstream is controlled by the nature of theagent, the nature of the linking group, the nature of the polymer, thesize of the polymer, the method of delivery or a combination of theforegoing.

In a fifteenth aspect, the present disclosure provides a method oftreatment for a disorder characterized by excessive GABA re-uptake orGABA re-uptake or an anxiety disorder, social anxiety disorder, panicdisorder, neuropathic pain, chronic pain, muscle tremors, muscle spasms,/seizures, convulsions and/or epilepsy, the method comprising the stepof administering a conjugate of the third-fourth, sixth or eighth-ninthaspects to a subject.

In a sixteenth aspect, the present disclosure provides a method oftreatment for a disorder characterized by excessive GABA re-uptake orGABA re-uptake or an anxiety disorder, social anxiety disorder, panicdisorder, neuropathic pain, chronic pain, muscle tremors, muscle spasms,seizures, convulsions and/or epilepsy, the method comprising the step ofadministering a conjugate of the third-fourth, sixth or eighth-ninthaspects to a subject, wherein the levels of the agents in thebloodstream is controlled by the nature of the agent, the nature of thelinking group, the nature of the polymer, the size of the polymer, themethod of delivery or a combination of the foregoing.

In any of the eleventh through sixteenth aspects, the conjugate isadministered to a subject by subcutaneous administration.

In any of the eleventh through sixteenth aspects, the levels of thereleased agent in the plasma of a subject is controlled by the dose ofPOZ-conjugate delivered via subcutaneous route.

In any of the eleventh through sixteenth aspects, the method oftreatment provides sustained, controllable delivery of the agent over aperiod of days to weeks.

In any of the eleventh through sixteenth aspects, the method oftreatment may further comprise identifying a subject in need of suchtreatment.

In any of the eleventh through sixteenth aspects, the conjugate isadministered in a therapeutically effective amount.

In a seventeenth aspect, the present disclosure provides for methods ofmanufacture of a conjugate of the first through tenth aspects.

In an eighteenth aspect, the present disclosure provides for kitscontaining a conjugate of the first through tenth aspects along withinstructions for administering the conjugate.

DETAILED DESCRIPTION Definitions

As used herein, the term “agent” refers to any molecule having atherapeutic or diagnostic application, wherein the agent is capable offorming a linkage with a functional group on a polymer or a linkinggroup attached to a polymer, the agent including, but not limited to, atherapeutic agent (such as but not limited to a drug), a diagnosticagent or an organic small molecule. In a specific embodiment, agent isuseful in the treatment of PD or other diseases or conditions related todopamine insufficiency in the peripheral or central nervous system. In aspecific embodiment, the agent is a dopamine agonist, adenosine A_(2A)antagonist, an anticholinergic, a monamine oxidase-B inhibitor or acatechol-O-methyl transferase (COMT) inhibitor. In a specificembodiment, the agent is useful in the treatment of a disordercharacterized by excessive GABA re-uptake or GABA re-uptake or ananxiety disorder, social anxiety disorder, panic disorder, neuropathicpain, chronic pain, muscle tremors, muscle spasms, seizures, convulsionsand/or epilepsy. In a specific embodiment, the agent is a dopamineagonist. In another specific embodiment, the agent is a GABA uptakeinhibitor.

As used herein, the term “link”, “linked” “linkage” or “linker” whenused with respect to a polymer or agent described herein, or componentsthereof, refers to groups or bonds that normally are formed as theresult of a chemical reaction and typically are covalent linkages.

As used herein, the term “releasable linker” or “releasablefunctionality” refers to a chemical linkage containing a cleavablemoiety that is cleavable in a subject in vivo under physiologicalconditions in the subject after a conjugate of the present disclosurehas been administered to the subject. In one embodiment, the cleavablemoiety is cleaved by a chemical reaction. In aspect of this embodiment,the cleavage is by reduction of an easily reduced group, such as, butnot limited to, a disulfide. In one embodiment, the cleavable moiety iscleaved by a substance that is naturally present or induced to bepresent in the subject. In an aspect of this embodiment, such asubstance is an enzyme or polypeptide. Therefore, in one embodiment, thecleavable moiety is cleaved by an enzymatic reaction. In one embodiment,the cleavable moiety is cleaved by a combination of the foregoing.

As used herein, the term “alkyl”, whether used alone or as part of asubstituent group, includes straight hydrocarbon groups comprising fromone to twenty carbon atoms. Thus the phrase includes straight chainalkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl,heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like. The phrasealso includes branched chain isomers of straight chain alkyl groups,including but not limited to, the following which are provided by way ofexample: —CH(CH₃)₂, —CH(CH₃)(CH₂CH₃), —CH(CH₂CH₃)₂, —C(CH₃)₃,—C(CH₂CH₃)₃, —CH₂ CH(CH₃)₂, —CH₂CH(CH₃)(CH₂CH₃), —CH₂CH(CH₂CH₃)₂,—CH₂C(CH₃)₃, —CH₂C(CH₂CH₃)₃, —CH(CH₃)CH(CH₃)(CH₂CH₃), —CH₂CH₂CH(CH₃)₂,—CH₂CH₂CH(CH₃)(CH₂CH₃), —CH₂CH₂CH(CH₂CH₃)₂, —CH₂CH₂C(CH₃)₃,—CH₂CH₂C(CH₂CH₃)₃, —CH(CH₃)CH₂CH(CH₃)₂, —CH(CH₃)CH(CH₃)CH(CH₃)CH(CH₃)₂,—CH(CH₂ CH₃)CH(CH₃)CH(CH₃)(CH₂CH₃), and others. The phrase also includescyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, and cyclooctyl and such rings substituted withstraight and branched chain alkyl groups as defined above. The phrasealso includes polycyclic alkyl groups such as, but not limited to,adamantyl norbornyl, and bicyclo[2.2.2]octyl and such rings substitutedwith straight and branched chain alkyl groups as defined above.

As used herein, the term “alkenyl”, whether used alone or as part of asubstituent group, includes an alkyl group having at least one doublebond between any two adjacent carbon atoms.

As used herein, the term “alkynyl”, whether used alone or as part of asubstituent group, includes an alkyl group having at least one triplebond between any two adjacent carbon atoms.

As used herein, the term “unsubstituted alkyl”, “unsubstituted alkenyl”and “unsubstituted alkynyl” refers to alkyl, alkenyl and alkynyl groupsthat do not contain heteroatoms.

As used herein, the term “substituted alkyl”, “substituted alkenyl” and“unsubstituted alkynyl” refers to alkyl alkenyl and alkynyl groups asdefined above in which one or more bonds to a carbon(s) or hydrogen(s)are replaced by a bond to non-hydrogen or non-carbon atoms such as, butnot limited to, an oxygen atom in groups such as alkoxy groups andaryloxy groups; a sulfur atom in groups such as, alkyl and aryl sulfidegroups, sulfone groups, sulfonyl groups, and sulfoxide groups; a siliconatom in groups such as in trialkylsilyl groups, dialkylarylsilyl groups,alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatomsin various other groups.

As used herein, the term “unsubstituted aralkyl” refers to unsubstitutedalkyl or alkenyl groups as defined above in which a hydrogen or carbonbond of the unsubstituted or substituted alkyl or alkenyl group isreplaced with a bond to a substituted or unsubstituted aryl group asdefined above. For example, methyl (CH3) is an unsubstituted alkylgroup. If a hydrogen atom of the methyl group is replaced by a bond to aphenyl group, such as if the carbon of the methyl were bonded to acarbon of benzene, then the compound is an unsubstituted aralkyl group(i.e., a benzyl group).

As used herein, the term “substituted aralkyl” has the same meaning withrespect to unsubstituted aralkyl groups that substituted aryl groups hadwith respect to unsubstituted aryl groups. However, a substitutedaralkyl group also includes groups in which a carbon or hydrogen bond ofthe alkyl part of the group is replaced by a bond to a non-carbon or anon-hydrogen atom.

As used herein, the term “unsubstituted aryl” refers to monocyclic orbicyclic aromatic hydrocarbon groups having 6 to 12 carbon atoms in thering portion, such as, but not limited to, phenyl, naphthyl,anthracenyl, biphenyl and diphenyl groups, that do not containheteroatoms. Although the phrase “unsubstituted aryl” includes groupscontaining condensed rings such as naphthalene, it does not include arylgroups that have other groups such as alkyl or halo groups bonded to oneof the ring members, as aryl groups such as tolyl are considered hereinto be substituted aryl groups as described below. Unsubstituted arylgroups may be bonded to one or more carbon atom(s), oxygen atom(s),nitrogen atom(s), and/or sulfur atom(s) in the, parent compound,however.

As used herein, the term “substituted aryl group” has the same meaningwith respect to unsubstituted aryl groups that substituted alkyl groupshad with respect to unsubstituted alkyl groups. However, a substitutedaryl group also includes aryl groups in which one of the aromaticcarbons is bonded to one of the non-carbon or non-hydrogen atoms, suchas, but not limited to, those atoms described above with respect to asubstituted alkyl, and also includes aryl groups in which one or morearomatic carbons of the aryl group is bonded to a substituted and/orunsubstituted alkyl, alkenyl, or alkynyl group as defined herein. Thisincludes bonding arrangements in which two carbon atoms of an aryl groupare bonded to two atoms of an alkyl or alkenyl, group to define a fusedring system (e.g. dihydronaphthyl or tetrahydronaphthyl). Thus, thephrase “substituted aryl” includes, but is not limited to tolyl, andhydroxyphenyl among others.

As used herein, the term “unsubstituted heterocyclyl” refers to botharomatic and nonaromatic ring compounds including monocyclic, bicyclic,and polycyclic ring compounds containing 3 or more ring members of whichone or more is a heteroatom such as, but not limited to, N, O, and S.Although the phrase “unsubstituted heterocyclyl” includes condensedheterocyclic rings such as benzimidazolyl, it does not includeheterocyclyl groups that have other groups such as alkyl or halo groupsbonded to one of the ring members, as compounds such as2-methylbenzimidazolyl are “substituted heterocyclyl” groups as definedbelow. Examples of heterocyclyl groups include, but are not limited to:unsaturated 3 to 8 membered rings containing 1 to 4 nitrogen atoms,condensed unsaturated heterocyclic groups containing 1 to 4 nitrogenatoms, unsaturated 3 to 8 membered rings containing 1 to 2 oxygen atomsand 1 to 3 nitrogen atoms, saturated 3 to 8 membered rings containing 1to 2 oxygen atoms and 1 to 3 nitrogen atoms such, unsaturated condensedheterocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogenatoms, unsaturated 3 to 8 membered rings containing 1 to 3 sulfur atomsand 1 to 3 nitrogen atoms, saturated 3 to 8 membered rings containing 1to 2 sulfur atoms and 1 to 3 nitrogen atoms, saturated and unsaturated 3to 8 membered rings containing 1 to 2 sulfur atoms, unsaturatedcondensed heterocyclic rings containing 1 to 2 sulfur atoms and 1 to 3nitrogen atoms, unsaturated 3 to 8 membered rings containing oxygenatoms, unsaturated condensed heterocyclic rings containing 1 to 2 oxygenatoms, unsaturated 3 to 8 membered rings containing an oxygen atom and 1to 2 sulfur atoms, saturated 3 to 8 membered rings containing 1 to 2oxygen atoms and 1 to 2 sulfur atoms, unsaturated condensed ringscontaining 1 to 2 sulfur atoms, and unsaturated condensed heterocyclicrings containing an oxygen atom and 1 to 2 oxygen atoms. Heterocyclylgroup also include those described above in which one or more S atoms inthe ring is double-bonded to one or two oxygen atoms (sulfoxides andsulfones).

As used herein, the term “substituted heterocyclyl” has the same meaningwith respect to unsubstituted heterocyclyl groups that substituted alkylgroups had with respect to unsubstituted alkyl groups. However, asubstituted heterocyclyl group also includes heterocyclyl groups inwhich one of the carbons is bonded to one of the non-carbon ornon-hydrogen atom, such as, but not limited to, those atoms describedabove with respect to a substituted alky and substituted aryl groups andalso includes heterocyclyl groups in which one or more carbons of theheterocyclyl group is bonded to a substituted and/or unsubstitutedalkyl, alkenyl or aryl group as defined herein. This includes bondingarrangements in which two carbon atoms of an heterocyclyl group arebonded to two atoms of an alkyl, alkenyl, or alkynyl group to define afused ring system. Examples, include, but are not limited to,2-methylbenzimidazolyl, 5-methylbenzimidazolyl, 5-chlorobenzthiazolyl,1-methyl piperazinyl, and 2-chloropyridyl among others.

As used herein, the term “unsubstituted heterocylalkyl” refers tounsubstituted alkyl or alkenyl groups as defined above in which ahydrogen or carbon bond of the unsubstituted alkyl or alkenyl group isreplaced with a bond to a substituted or unsubstituted heterocyclylgroup as defined above. For example, methyl (CH₃) is an unsubstitutedalkyl group. If a hydrogen atom of the methyl group is replaced by abond to a heterocyclyl group, such as if the carbon of the methyl werebonded to carbon 2 of pyridine (one of the carbons bonded to the N ofthe pyridine) or carbons 3 or 4 of the pyridine, then the compound is anunsubstituted heterocyclylalkyl group.

As used herein, the tem' “substituted heterocylalkyl” has the samemeaning with respect to unsubstituted heterocyclylalkyl groups thatsubstituted aryl groups had with respect to unsubstituted aryl groups.However, a substituted heterocyclylalkyl group also includes groups inwhich a non-hydrogen atom is bonded to a heteroatom in the heterocyclylgroup of the heterocyclylalkyl group such as, but not limited to, anitrogen atom in the piperidine ring of a piperidinylalkyl group.

As used herein, the terms “treatment”, “treat” and “treating” refers acourse of action (such as administering a conjugate or pharmaceuticalcomposition) initiated after the onset of a symptom, aspect, orcharacteristics of a disease or condition so as to eliminate or reducesuch symptom, aspect, or characteristics. Such treating need not beabsolute to be useful.

As used herein, the term “in need of treatment” refers to a judgmentmade by a caregiver that a patient requires or will benefit fromtreatment. This judgment is made based on a variety of factors that arein the realm of a caregiver's expertise, but that includes the knowledgethat the patient is ill, or will be ill, as the result of a disease orcondition that is treatable by a method or compound of the disclosure.

As used herein, the term “in need of prevention” refers to a judgmentmade by a caregiver that a patient requires or will benefit fromprevention. This judgment is made based on a variety of factors that arein the realm of a caregiver's expertise, but that includes the knowledgethat the patient will be ill or may become ill, as the result of adisease or condition that is preventable by a method or compound of thedisclosure.

As used herein, the term “individual”, “subject” or “patient” refers toany animal, including mammals, such as mice, rats, other rodents,rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, andhumans. The term may specify male or female or both, or exclude male orfemale.

As used herein, the term “therapeutically effective amount” refers to anamount of a conjugate, either alone or as a part of a pharmaceuticalcomposition, that is capable of having any detectable, positive effecton any symptom, aspect, or characteristics of a disease or condition.Such effect need not be absolute to be beneficial.

General Description

The present disclosure provides polymer conjugates consisting of,consisting essentially of or comprising a water-soluble polymer and anagent. In one embodiment, the agent may be linked to the polymerbackbone via a direct linkage through a reactive group on the agent anda reactive group on the polymer. In one embodiment, the direct linkagecontains at least one cleavable moiety such that in vivo underphysiological conditions in the body of a subject, such as, but notlimited to, a human, the agent is released from the polymer at somepoint after administration of the polymer conjugate to the subject. Inan alternate embodiment, the agent may be linked to the polymer througha linking group. In one embodiment, the linking group contains at leastone cleavable moiety such that in vivo under physiological conditions inthe body of a subject, such as, but not limited to, a human, the agentis released from the polymer at some point after administration of thepolymer conjugate to the subject. Such releasable moieties are discussedherein. In one embodiment, the linking group contains, in addition tothe cleavable moiety, a group capable of forming a linkage with areactive group on the polymer, and a group capable of forming a linkagewith a reactive group on the agent. Regardless of the form of thelinkage, the linkage is a releasable linkage that allows the agent to bereleased from the polymer at some point after administration of theconjugate to a subject via cleavage of the cleavable moiety. The releasekinetics of the agent from the conjugate provides sustained,controllable delivery of the agent over a period of days to weeks. Inone embodiment, the release kinetics of the agent from the polymer iscontrolled by the nature of the linking group, the nature of the agent,the nature of the polymer, the size of the polymer, the method ofdelivery or a combination of the foregoing. In one embodiment, therelease kinetics of the agent from the polymer is controlled by thenature of the linking group. In one embodiment, the release kinetics ofthe agent from the polymer is controlled by the nature of the linkinggroup and/or the nature of the agent. In one embodiment, the releasekinetics of the agent from the polymer is controlled by the nature ofthe linking group and/or the nature of the polymer. In one embodiment,the release kinetics of the agent from the polymer is controlled by thenature of the linking group, the nature of the agent and/or the natureof the polymer.

In a general embodiment, the polymer conjugate of the present disclosuremay be represented by the general formula I.

POL_(n)-L-A_(b)   I

wherein,POL is a water-soluble polymer;n is 1-1000 and represent the number of monomer units comprising thewater-soluble polymer;b is 1 to 50, provided that n is always greater than or equal to b;L is an optional linking group containing a cleavable moiety orrepresents a direct linkage through a reactive group on the agent and areactive group on the polymer, provided that the direct linkage forms acleavable moiety; andA is an agent.

The polymer portion of the disclosed polymer conjugates may take on avariety of forms. In certain embodiments, the polymer is apoly(oxazoline) (POZ), poly(5,6-dihydro-4h-1,3-oxazine), a dextran, adextran modified by oxidation, a polyethylene glycol (PEG), apoly(hydroxypropylmethacrylate), a polyglutamic acid, apolylactic-polyglutamic acid mixture, a polysialic acid, apolycaprolactone, a polyvinylpyrrolidone, a glycosaminoglycans, apolyglycerol, a poly(acryloyloxyethylphosphorylcholine), or amethacrylate-based copolymer with synthetic forms of phosphorylcholine;combinations of the foregoing are also included.

In one embodiment, the polymer is a poly(oxazoline) (POZ). In stillanother embodiment, the polymer is a polyethylene glycol (PEG). In stillanother embodiment, the polymer is a dextran. In still anotherembodiment, the polymer is a dextran modified by oxidation.

The agent may be any agent useful in the treatment of a disease orcondition or the diagnosis of a disease or condition. In certainembodiments, the agent is a diagnostic agent or a therapeutic agent. Incertain embodiment, the therapeutic agent is an organic small molecule.In one embodiment, the agent is a compound useful in the treatment of PDor other diseases or conditions related to dopamine insufficiency in theperipheral or central nervous system. In another embodiment, the agentis useful in the treatment of a disorder characterized by excessive GABAre-uptake or GABA re-uptake. In another embodiment, the agent is usefulin the treatment of an anxiety disorder, social anxiety disorder, panicdisorder, neuropathic pain, chronic pain, muscle tremors, muscle spasms,seizures, convulsions and/or epilepsy. The nature of the agents isdescribed in more detail in the present disclosure.

The linking group may form linkages with any reactive group on thepolymer backbone and any reactive group on the agent. The linkagebetween the linking group and the polymer may be formed on a terminalend of the polymer. Alternatively, the linkage between the linking groupand the polymer may be formed using a side chain group of the polymer(referred to herein as a “pendent” position). Furthermore, the linkinggroup may include components of the reactive group that was originallypresent on the polymer or the agent.

Suitable linking groups are described herein. In a particularembodiment, the polymer conjugates of the present disclosure may berepresented by the general formula II.

R-POZ_(n)-L-A_(b)   II

wherein,R is an initiating group;POZ is a polyoxazoline polymer;n is 1-1100 and represent the number of monomer units comprising thepolyoxazoline polymer;b is 1 to 50, provided that n is always greater than or equal to b;L is an optional linking group containing a cleavable moiety orrepresents a direct linkage through a reactive group on the agent and areactive group on the polymer, provided that the direct linkage forms acleavable moiety; and A is an agent.

A variety of POZ polymers may be used in the POZ conjugates of thepresent disclosure. The POZ may contain a single type or class offunctional groups or may contain more than one type or class offunctional groups. The POZ be a linear POZ polymer, a branched POZpolymer, a pendent POZ polymer or a multi-armed POZ polymer. Variousrepresentative POZ polymers are described herein. The POZ polymer may beprepared by living cation polymerization or by other methods as is knownin the art. Representative POZ polymers are described in U.S. Pat. Nos.7,943,141, 8,088,884, 8,110,651 and 8,101,706, application Ser. Nos.13/003,306, 13/549,312 and 13/524,994, each of which is incorporatedherein by reference for such teachings. In one embodiment, the POZpolymer is prepared by living cation polymerization.

The agent may be any agent useful in the treatment of a disease orcondition or the diagnosis of a disease or condition. In certainembodiments, the agent is a diagnostic agent or a therapeutic agent. Incertain embodiment, the therapeutic agent is an organic small molecule.In one embodiment, the agent is a compound useful in the treatment of PDor other diseases or conditions related to dopamine insufficiency in theperipheral or central nervous system. In another embodiment, the agentis useful in the treatment of a disorder characterized by excessive GABAre-uptake or GABA re-uptake. In another embodiment, the agent is usefulin the treatment of an anxiety disorder, social anxiety disorder, panicdisorder, neuropathic pain, chronic pain, muscle tremors, muscle spasms,seizures, convulsions and/or epilepsy. The nature of the agents isdescribed in more detail in the present disclosure.

In one embodiment, the POZ polymer contains at least one reactive groupcapable of forming a linkage with an agent or a linking group.

The linkage (whether a direct linkage or a linkage utilizing a linkinggroup) between the polymer and agent may be formed between any reactivegroup on the polymer backbone and any reactive group on the agent. Thelinkage between the linking group and the polymer may be formed on aterminal end of the polymer. Alternatively, the linkage between thelinking group and the polymer may be formed using a side chain group ofthe polymer (referred to herein as a “pendent” position). Furthermore,the linkage (whether a direct linkage or a linkage utilizing a linkinggroup) may include components of the reactive group that was originallypresent on the polymer or the agent. Suitable linking groups aredescribed herein.

Exemplary R groups include, but are not limited to, hydrogen, alkyl andsubstituted alkyl. In one embodiment, the initiating group is an alkylgroup, such as a C1 to C4 alkyl group. In a specific embodiment of theforegoing, the initiating group is a methyl group. In anotherembodiment, the initiating group is H. In yet another embodiment, theinitiating group is selected to lack a functional group. Additionalexemplary initiating groups are disclosed in U.S. Pat. Nos. 7,943,141,8,088,884, 8,110,651 and 8,101,706, application Ser. Nos. 13/003,306,13/549,312 and 13/524,994, each of which is incorporated herein byreference for such teachings.

In a particular embodiment, the POZ conjugate of the present disclosuremay be represented by the general formula IIA, wherein the linkagebetween the agent and the polymer is formed at the “pendent” position.

whereinR, POZ, n, b, L and A are as defined in the description of formula II;andT is a terminating group.

In one embodiment, T is a terminating nucleophile. In one embodiment, Tis Z-B-Q, wherein Z is S, O, or N; B is an optional linking group; and Qis a terminating nucleophile or a terminating portion of a nucleophile.In certain embodiments Q is inert (i.e., does not contain a functionalgroup); in other embodiments, Q contains a second functional group.

Exemplary B groups include, but are not limited to, alkylene groups. Ina particular embodiment, B is —(CH₂)_(y)— where y is an integer selectedfrom 1 to 16. In a particular embodiment, Z is S. POZ conjugatescontaining a sulfur group as described herein may be prepared byterminating the POZ cation with a mercaptide reagent, such as, but notlimited to, a mercapto-ester (for example, —S—CH₂CH₂—CO₂CH₃) ormercapto-protected amine (for example, —S—CH₂CH₂—NH— tBoc). Such POZconjugates provide for effective, large-scale purification byion-exchange chromatography (to remove secondary amines), as well asallowing for control of polydispersity values (with polydispersityvalues of 1.10 or less) and for the creating of conjugates with highermolecular weight POZ polymers. In another embodiment, Z is N. In afurther embodiment, Z is O.

As stated above, Q may be inert or may contain a functional group. WhenQ contains a functional group, exemplary groups include, but are notlimited to, alkyne, alkene, amine, oxyamine, aldehyde, ketone, acetal,thiol, ketal, maleimide, ester, carboxylic acid, activated carboxylicacid (such as, but not limited to, N-hydroxysuccinimidyl (NHS) and1-benzotriazine active ester), an active carbonate, a chloroformate,alcohol, azide, vinyl sulfone, or orthopyridyl disulfide (OPSS). When Qis an inert group, any inert group may be used, including, but notlimited to —C₆H₅.

In one embodiment, L is present and contains a cleavable moiety, Z is S,B is —CH₂CH₂— and Q is —COOH. In another specific embodiment L ispresent and contains a cleavable moiety, Z is O, B is —CH₂CH₂— and Q is—COOH. In still another specific embodiment L is present and contains acleavable moiety, Z is N, B is —CH₂CH₂— and Q is —COOH.

In another particular embodiment, the POZ conjugate of the presentdisclosure may be represented by the general formula IIB, wherein thelinkage between the agent and the polymer is formed at the “pendent”position.

whereinR, L, A are as defined in the description of formula II and T (includingthe definitions of Z, B and Q) is as described in the description offormula IIA;R₁ is a non-reactive group;a is ran which indicates a random copolymer or block which indicates ablock copolymero is an integer from 1 to 50; andm is an integer from 1 to 1000.

In one embodiment, R₁ is an alkyl or a substituted alkyl. In aparticular embodiment, R₁ is methyl, ethyl, propyl or butyl. ExemplaryR₁ groups are described in U.S. Pat. Nos. 7,943,141, 8,088,884,8,110,651 and 8,101,706, application Ser. Nos. 13/003,306, 13/549,312and 13/524,994, each of which is incorporated herein by reference forsuch teachings.

In a particular embodiment, T is Z-B-Q and the compound is representedby the general formula IIC.

whereinR, L, A are as defined in the description of formula II and Z, B and Qare as described in the description of formula IIA and R₁ is as definedin the description for the formula IIB;

In one embodiment, L is present and contains a cleavable moiety, Z is S,B is —CH₂CH₂— and Q is —COOH. In another specific embodiment L ispresent and contains a cleavable moiety, Z is O, B is —CH₂CH₂— and Q is—COOH. In still another specific embodiment L is present and contains acleavable moiety, Z is N, B is —CH₂CH₂— and Q is —COOH.

In one embodiment of the conjugates of formula IIB and IIC, the POZconjugate is formed by reacting a POZ polymer of the general formulaR-{[N(COX)CH₂CH₂]_(o)—[N(COR₁)CH₂CH₂]_(m)}_(a)- with an agent or alinking group. In the general formula above, X represents a pendentmoiety containing a functional group capable of forming a linkage withan agent or a linking group. As a result of the linkage being formed,the COX portion of the POZ polymer becomes a part of the linkage linkingthe polymer and the agent. Exemplary functional groups for X include,but are not limited to, alkene, alkyne, aralkyl, heterocycloalkyl,amine, oxyamine, aldehyde, ketone, acetal, ketal, maleimide, ester,carboxylic acid, activated carboxylic acid (such as, but not limited to,N-hydroxysuccinimidyl (NHS) and 1-benzotriazine active ester), an activecarbonate, a chloroformate, alcohol, azide, vinyl sulfone, ororthopyridyl disulfide (OPSS). X may comprise a linking portion thatlinks the functional group to the polyoxazoline polymer. Exemplarylinking portions include alkylene groups. In certain cases, the alkylenegroup is a C₁-C₁₅ alkylene group.

In a particular embodiment, X contains an alkyne group and the agent orlinking group contains an azido group. In another embodiment, X containsan azido group and the agent or linking group contains an alkyne group.In still a further embodiment, X contains a carboxylic acid group andthe linking group contains a phenolic group.

In the embodiments shown in FIGS. IIB and IIC, the number of agents andlinking groups attached to the polymer conjugate is defined by thevariable o as this polymer block contains the pendent moiety containinga functional group capable of forming a linkage with an agent or alinking group. In one embodiment, the number of agents and linkinggroups attached to the polymer conjugate is equal to the value of thevariable o. In another embodiment, the number of agents and linkinggroups attached to the polymer conjugate is less than the value of thevariable o.

In the embodiments described above for the general formulas I, II, IIA,IIB and IIC, specific linking groups are as described below. For thesake of clarity any linking group described herein may be used in thegeneral formulas described above.

Linking Group

In the embodiments described above, the agent is linked to the polymervia a releasable linkage. In one embodiment, a linking group is providedbetween the polymer and the agent, the linking group containing acleavable moiety. The linking group is capable of forming a releasablelinkage between the polymer and the agent. In other words the linkinggroup contains a linkage that can be cleaved in vivo in a subject afteradministration of a polymer conjugate of the present disclosure to thesubject. In one embodiment, the cleavable moiety is cleaved by achemical reaction. In aspect of this embodiment, the cleavage is byreduction of an easily reduced group, such as, but not limited to, adisulfide. In one embodiment, the cleavable moiety is cleaved by asubstance that is naturally present or induced to be present in thesubject. In an aspect of this embodiment, such a substance is an enzymeor polypeptide. Therefore, in one embodiment, the cleavable moiety iscleaved by an enzymatic reaction. In one embodiment, the cleavablemoiety is cleaved by a combination of the foregoing. The linking groupmay contain portions of the polymer and/or portions of the agent as suchportions have reacted to form the linking group as discussed below.

Exemplary releasable moieties include, but are not limited to, esters,carboxylate esters (—C(O)—O—), carbonate esters (—O—C(O)—O—), carbamates(—O—C(O)—NH—) and amides (—C(O)—NH—); other releasable moieties arediscussed herein. In a particular embodiment, the cleavable moiety is anester. In another particular embodiment, the cleavable moiety is acarbonate ester or a carboxylate ester. In addition, the linking groupmay be a naturally occurring amino acid, a non-naturally occurring aminoacid or a polymer containing one or more naturally occurring and/ornon-naturally occurring amino acids. The linking group may includecertain groups from the polymer chain and/or the agent.

In the descriptions below, the polymer is assumed to be a polyoxazolinepolymer for the purpose of exemplification. However, the reactions beloware equally applicable to other polymer types.

In one embodiment, the linking group is a di-substituted triazole thatcontains a cleavable moiety in one of the R₃ or R₄ groups. In oneembodiment, the cleavable moiety is present in the R₄ group. In aspecific embodiment, the di-substituted triazole has the structure:

In another embodiment, the di-substituted triazole has the structure:

In each of the foregoing structures:R₃ is a linker linking the triazole moiety to the polymer chain. R₃ maybe defined in part by the functional group on the polymer chain; inother words, R₃ may contain a part of the functional group on thepolymer chain. In one embodiment, R₃ is —C(O)—R₅—, where R₅ is absent oris a substituted or unsubstituted alkyl from 1 to 10 carbons in length.R₄ is a linker linking the triazole moiety to the agent. R₄ may bedefined in part by the functional group on the agent; in other words, R₄may contain a part of the functional group on the agent. In oneembodiment, R₄ is —R₆—R₇—R₈—, where R₆ is a substituted or unsubstitutedalkyl, substituted or unsubstituted aralkyl or a oligo(ethylene oxide)(for example, —(CH₂CH₂O)_(d)— where d is 1-10 or 1-4), R₇ is a groupcontaining the cleavable moiety or a portion of cleavable moiety and R₈is absent or O, S, CR_(c), or NR_(c), where R_(c) is H or substituted orunsubstituted alkyl. In certain embodiments, R₇ and R₈ may combine toform the cleavable moiety. In one embodiment, R₇ is —R_(a)—(O)—R_(b)—,—R_(a)—O—C(O)—R_(b)—, —R_(a)—C(O)—NH— cyclic-O—C(O)—R_(b)— (where cyclicrepresents substituted or unsubstituted aryl, heterocylalkyl,heterocycle or cycloalkyl), —R_(a)—C(O)—NH—(C₆H₄)—O—C(O)—R_(b)—,—R_(a)—C(O)—R_(b)—, —R_(a)—C(O)—O—R_(b)—, —R_(a)—O—C(O)—O—R_(b)—,—R_(a)—O—C(O)—NR₁₅—R_(b)— (where R₁₅ is a is H or a substituted orunsubstituted C1-C5 alkyl), —R_(a)—CH(OH)—O—R_(b)—, —R_(a)—S—S—R_(b)—,—R_(a)—O—P(O)(OR₁₁)—O—R_(b)— (where R₁₁ is H or a substituted orunsubstituted C₁-C5 alkyl), or —R_(a)—C(O)—NR₁₅—R_(b)— (where R₁₅ is ais H or a substituted or unsubstituted C1-C5 alkyl), where R_(a) andR_(b) are each independently absent or substituted or unsubstitutedalkyl. In another embodiment, R_(a) and R_(b) are each independentlyabsent or a C2-C16 substituted or unsubstituted alkyl. In one embodimentof the foregoing, R₆ is a straight chain substituted or unsubstitutedC1-C16 alkyl or a branched substituted or unsubstituted C1-C16 alkyl, R7is —R_(a)—C(O)—O—R_(b)— and R₈ is absent. In one embodiment of theforegoing, R₆ is a straight chain substituted or unsubstituted C1-C4alkyl or a branched substituted or unsubstituted C1-C4 alkyl, R₇ is—R_(a)—C(O)—O—R_(b)— and R8 is absent. In one embodiment of theforegoing, R₆ is, —CH₂—, —CH₂—CH₂—, or —CH₂(CH₃)— and R₇ is —C(O)—O— andR₈ is absent.

In a particular embodiment, R₃ is —C(O)—(CH₂)₃ and R₄ is —CH₂—C(O)—O—,—CH₂—CH₂—C(O)—O— or —CH₂(CH₃)—C(O)—O—.

In a particular embodiment, R₃ is —C(O)—(CH₂)₃ and R₄ is—CH₂—CH₂—O—C(O), —CH₂—CH₂—CH₂—O—C(O), —CH₂—CH₂—CO—NH—(C₆H₄)—O—C(O)— or—(CH₂CH₂O)_(d)—C(O)—, where d is 1-10.

In another embodiment, the linking group has the structure R₉—Y—R₁₀,where Y is a cleavable moiety and R₉ and R₁₀ are each groups linking Yto the polymer conjugate and the agent, respectively. R9 and R₁₀ may bethe same of different. In one embodiment, R₉ and R₁₀ are eachindependently absent or substituted or unsubstituted alkyl, substitutedor unsubstituted aralkyl or a oligo(ethylene oxide) (for example,—(CH₂CH₂O)_(d)— where d is 1-10 or 1-4). In another embodiment, R₉ andR₁₀ are each independently absent or a C2-C16 substituted orunsubstituted alkyl.

In one embodiment of the foregoing, the linking group Y is R₉—(O)—R₁₀—,—R₉—O—C(O)—R₁₀—, —R9—C(O)—NH-cyclic-O—C(O)—R₁₀— (where cyclic representssubstituted or unsubstituted aryl, heterocylalkyl, heterocycle orcycloalkyl), —R₉—C(O)—NH—(C₆H₄)—O—C(O)—R₁₀—, —R₉—C(O)—R₁₀—,—R₉—C(O)—O—R₁₀—, —R₉—O—C(O)—O—R₁₀—, —R₉—O—C(O)—NR₁₆—R₁₀— (where R₁₆ is ais H or a substituted or unsubstituted C1-C5 alkyl), —R₉—CH(OH)—O—R₁₀—,—R₉—S—S—R₁₀—, —R₉—O—P(O)(OR₁₂)—O—R₁₀— (where R₁₂ is H or a substitutedor unsubstituted C1-C5 alkyl), —R₉—C(O)—NR₁₆—R₁₀— (where R₁₆ is a is Hor a substituted or unsubstituted C1-C5 alkyl) or—R₉—[NR₁₆—CH(R₁₃)(R₁₄)—C(O)]_(q)—R₁₀— (where R₁₆ is a is H or asubstituted or unsubstituted C1-C5 alkyl, R₁₃ is H or a C1-C5 alkyl, R₁₄is a side chain group on a naturally occurring or non-naturallyoccurring amino acid and q is 1-10), where R₉ and R₁₀ are eachindependently absent or substituted or unsubstituted alkyl. In anotherembodiment, R₉ and R₁₀ are each independently absent, a C1-C16 or aC1-C4 substituted or unsubstituted alkyl.

In one embodiment, the release kinetics of the agent from the polymer iscontrolled by the nature of the linking group, the nature of the agent,the nature of the polymer, the size of the polymer, the method ofdelivery or a combination of the foregoing. In one embodiment, therelease kinetics of the agent from the polymer is controlled by thenature of the linking group. In one embodiment, the release kinetics ofthe agent from the polymer is controlled by the nature of the linkinggroup and/or the nature of the agent. In one embodiment, the releasekinetics of the agent from the polymer is controlled by the nature ofthe linking group and/or the nature of the polymer. In one embodiment,the release kinetics of the agent from the polymer is controlled by thenature of the linking group, the nature of the agent and/or the natureof the polymer. Furthermore, diffusion of the free agent can also play arole.

In each of the foregoing, the cleavable moiety may be cleaved chemicallyunder physiological conditions, cleaved by a substance that is naturallypresent or induced to be present in the subject under physiologicalconditions or by a combination of the foregoing. In one embodiment, suchsubstance is an enzyme or polypeptide and the cleavage is an enzymaticcleavage.

Agent

The agent may be any agent useful in the treatment of a disease orcondition or the diagnosis of a disease or condition. In certainembodiments, the agent is a diagnostic agent or a therapeutic agent. Incertain embodiment, the therapeutic agent is an organic small molecule.Furthermore, the agent may be any molecule having a therapeutic ordiagnostic application, wherein the agent is capable of forming alinkage with a functional group on a polymer of the present disclosure,such as but not limited to, a POZ polymer, or a linking group linked toa polymer of the present disclosure.

In one embodiment, the agent is useful for the treatment of PD or otherdiseases or conditions related to dopamine insufficiency in theperipheral or central nervous system. In such an embodiment, the agentmay be a dopamine agonists, dopamine antagonist, adenosine A_(2A)receptor antagonists, anticholinergics, monamine oxidase-B inhibitorsand catechol-O-methyl transferase (COMT) inhibitors. Exemplary dopamineagonists include, but are not limited to, rotigotine, pramipexole,quinagolide, fenoldopam, apomorphine, 5-OH-DPAT, ropinirole, pergolide,cabergoline, and bromocriptine. Exemplary anticholinergics include, butare not limited to, trihexyphenidyl, biperidin and hyoscyamine.Exemplary monamine oxidase-B inhibitors include, but are not limited to,seligiline and rasagiline. Exemplary COMT inhibitors include, but arenot limited to, tolcapone and entacapone. Exemplary Adenosine A_(2A)receptor antagonists include, but not limited to, caffeine,theophylline, istradefylline, and preladenant (B. C. Cook and P. F.Jackson, Adenosine A_(2A) receptor antagonists and Parkinson's disease,ACS Chemical Neuroscience, 2011, 2, 555-567).

PD is a central nervous system disorder resulting from loss of dopamineneurons in the substantia nigra pars compacta. The loss of these neuronsin the brain leads to a deficiency of dopamine, a neurotransmitter thatis essential for normal coordination and movement. Striatal dopaminergicneurons fire in a random, but continuous fashion due to stable levels ofdopamine, allowing for precisely coordinated movements. In PD patientsthe pre-synaptic neurons degenerate. Administration of dopaminergicagents (dopamine agonists and levo-dopa) in an attempt to controlsymptoms leads to discontinuous stimulation of the post-synapticneurons, promoting motor fluctuations that can worsen as the diseaseprogresses (dyskinesias). Early symptoms of dopamine deficiency in PDinclude tremors, rigidity, bradykinesia, and gait problems. Cognitiveand behavioral problems as well as dementia occur in later stages of PD.

While there is no cure, for PD at this time, symptoms of this diseaseare treated with a variety of drugs aimed at maintaining dopaminergictone. Drugs currently used for the treatment of PD include levodopa,dopamine agonists, adenosine A_(2A) antagonist, anticholinergics,monamine oxidase-B inhibitors and catechol-O-methyl transferaseinhibitors and other drugs. Levodopa is typically reserved for the laterstages of PD while the other classes are the drugs of choice in theearly stages of PD. There are challenges associated with these drugs.Levodopa can be administered orally, but gastrointestinal tractmetabolism and erratic absorption limit bioavailability. For levodopa,bioavailability is less than 10% and even less reaches the brain intactdue to peripheral metabolism, including metabolism by decarboxylaseenzymes. To address this issue, decarboxylase inhibitors such ascarbidopa are co-administered to inhibit peripheral metabolism.Furthermore, the short half-lives of these drugs require frequent dosingof several times daily which results in pulsatile stimulation ofstriatal dopamine receptors; this may actually accelerate the demise ofdopaminergic neurons in the CNS. Low solubility of some of thesecompounds, with limited oral bioavailabity, further complicates theirclinical use.

The use of dopamine agonists to treat PD is known in the art. The useof, 2-aminotetralins (a class of compounds with dopamine agonistactivity) date back to the late 1980s in disclosures by Horn, A. S.(U.S. Pat. No. 4,722,933, February 1988 and U.S. Pat. No. 4,885,308,December 1989). Horn discussed analogues and small molecule pro-drugs of2-aminotetralin to treat central nervous system disorders. One suchexample is rotigotine, a potent dopamine agonist. However,administration of rotigotine has proven to be difficult due to poorsolubility in aqueous medium and short half-life. Swart and de Zeeuwreport that oral and intraperitoneal bioavailability of rotigotine inrats to be less than 10% (Pharmacokinetics of the dopamine D2 agonistS(-)-2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin in freelymoving rats. J. Pharm. Sci. 1993 February; 82(2):200-3). Studies in manshow that rotigotine has a half-life of 2.5 hours and it is rapidlymetabolized to the sulfate and glucuronide analogues at the phenolicgroup. In an effort to improve the characteristics and oralbioavailability of these dopamine agonists, Stefano, Sozio, and Cerasa(Molecules 2008, 13: 46-68) prepared acetyl, propionyl, isobutyryl andcarbamate pro-drugs. Esters of this type, however, would not be expectedto improve water solubility and the improvement in duration in actionwas marginally increased from 3 to 4 hours to 11 to 15 hours. Atransdermal patch was developed to address the suboptimalpharmacokinetics. This approach allows for 24 hours of delivery andimproved bioavailability, but stability issues relating to poorsolubility and crystallization in the patch resulted in this product'swithdrawal from the U.S. market until formulation issues were addressed.

Ropinirole is another non-ergoline dopamine agonist that is deliveredorally and has a half-life of 3 to 6 hours in man. Higher doses arerequired to achieve clinical benefit due to hepatic and renalmetabolism. In addition, the once-a-day tablet dose generates undesiredpeak and troughs in blood concentration.

In another embodiment, the agent is useful for the treatment of adisorder characterized by excessive GABA re-uptake or GABA re-uptake. Inone embodiment, the agent is useful in the treatment of an anxietydisorder, social anxiety disorder, panic disorder, neuropathic pain(which includes usefulness in poorly understood disorders likefibromyalgia), chronic pain, muscle tremors, muscle spasms, seizures,convulsions and/or epilepsy. In such an embodiment, the agent may be aGABA re-uptake inhibitor. GABA (gamma-aminobutyric acid) is aneurotransmitter produced in the central nervous system that is thoughtto be the major inhibitory neurotransmitter. Inhibition of its re-uptakeby certain small molecules (for example, tiagabine and nipecotic acid)potentiate its activity in the post-synaptic neuron and potentiateGABAergic neurotransmission.

Therefore, there is a need in the art for new compositions for thetreatment of PD and other conditions relating to dopamine deficiency aswell as for the treatment of an anxiety disorder, social anxietydisorder, panic disorder, neuropathic pain (which includes usefulness inpoorly understood disorders like fibromyalgia), chronic pain, muscletremors, muscle spasms, seizures, convulsions and/or epilepsy.

The present disclosure provides conjugates containing a polymer, such asthose described herein, and an agent useful in the treatment of PD orother diseases or conditions related to dopamine insufficiency in theperipheral or central nervous system as well as the treatment of anxietydisorders, social anxiety disorders, panic disorders, neuropathic pain(which includes usefulness in poorly understood disorders likefibromyalgia), chronic pain, muscle tremors, muscle spasms, seizures,convulsions and/or epilepsy. The foregoing disorders will benefit from apolymer approach for sustained pharmacokinetics, increasedbioavailability and ease of administration.

The polymer conjugates of the present disclosure have been exemplifiedby POZ-rotigotine, POZ-tiagabine, POZ-ropinirole, PEG-rotigotine,PEG-tiagabine, and dextran-rotigotine. Other agents and polymers,including those disclosed herein, are also useful in the conjugates ofthe present disclosure provided such agents and polymers have, or can bemodified to contain, appropriate functionality for linkage to the watersoluble polymer.

Dopamine Agonists

Other classes of drugs useful in the treatment of PD, such as, but notlimited to, anticholinergics (such as, but not limited to,trihexyphenidyl, biperidin and hyoscyamine), monamine oxidase-Binhibitors (such as, but not limited to, seligiline and rasagiline),catechol-O-methyl transferase (COMT) inhibitors (such as, but notlimited to, tolcapone and entacapone) and adenosine A_(2A) receptorantagonists (such as, but not limited to, preladenant, theophylline andistradefylline) are also useful in the conjugates and methods oftreatment described herein.

For clarity, the agent may be any of the foregoing classes of compoundsor a compound of another class that have appropriate chemicalfunctionality to form a releasable linkage with a water-soluble polymeror linking group of the present disclosure. The foregoing examples arepresented by way of exemplification and are not intended to be limiting.

Furthermore, the agent may be used to treat a variety of diseases orconditions. The present specification described certain agents usefulfor the treatment of PD and other diseases or conditions related todopamine insufficiency in the peripheral or central nervous system andagents useful for the treatment of anxiety disorders, social anxietydisorders, panic disorders, neuropathic pain (which includes usefulnessin poorly understood disorders like fibromyalgia), chronic pain, muscletremors, muscle spasms, seizures, convulsions and/or epilepsy in orderto illustrate the teachings of the present disclosure. However, thechoice of agent should not be limited to the treatment of theexemplified diseases or conditions. Any agent that would benefit from apolymer approach for sustained pharmacokinetics, increasedbioavailability and ease of administration may also be used. Theforegoing examples are presented by way of exemplification and are notintended to be limiting.

Control of Release of Agent

The present disclosure provides polymer conjugates where the releasekinetics of the agent from the water-soluble polymer can be controlledby varying one or more parameters of the polymer conjugate. Suchparameters include, but are not limited to, the nature of the linkinggroup, the nature of the polymer, the nature of the agent, the size ofthe polymer, and varying the method of delivery (mode ofadministration). Tables 1-4 provide experimental data on control ofcleavage rates by varying the nature of the linker, drug and polymer.

In one embodiment, the release kinetics of the agent from thewater-soluble polymer is controlled by the nature of the linking group.In another embodiment, the release kinetics of the agent from thewater-soluble polymer is controlled by the nature of the polymer. Inanother embodiment, the release kinetics of the agent from thewater-soluble polymer is controlled by the nature of the agent. Inanother embodiment, the release kinetics of the agent from thewater-soluble polymer is controlled by the size of the polymer. Inanother embodiment, the release kinetics of the agent from thewater-soluble polymer is controlled by the mode of administration. Instill a further embodiment, the release kinetics of the agent from thewater-soluble polymer is controlled by the nature of the linking groupand/or the nature of the agent. In still a further embodiment, therelease kinetics of the agent from the water-soluble polymer iscontrolled by the nature of the linking group and/or the nature of thepolymer. In still a further embodiment, the release kinetics of theagent from the water-soluble polymer is controlled by the nature of thelinking group, the nature of the agent and/or the nature of the polymer.

As discussed above, the release kinetics of the agent from thewater-soluble polymer (i.e., rate of cleavage of the linking group) iscontrolled, in one embodiment, by the nature of the linking group. Forexample, as shown in Table 1 for cleavage of polymer-triazine-alkyl-CO₂—rotigotine, changes in the alkyl group affect the release of the drugrotigotine. Similarly, the nature of the polymer has an effect on therelease kinetics of the agent from the water-soluble polymer. Forexample, rotigotine is released much more slowly from POZ than from PEGor modified dextran (Table 1). Slower release of the agent avoids arapid spike in drug concentration in the blood followed by rapidclearance. Such a profile results in sustained release of drug overtime. In some instances a single administration of a polymer conjugateof the present disclosure can provide for therapeutically effectiveconcentrations of the agent in the blood over a period of several daysto weeks.

Table 2 illustrates that rate of release of an agent from a polymerconjugate of the present disclosure is affected by the drug itself.Variation ,of polymer and linker can be used to tune the release rate ofeach agent within a certain range determined by the agent. Table 3illustrates that varying the molecular weight of polymer and the numberof pendents has no effect on rate of release of the agent (irinotecan inthis case) from the polymer.

In addition, the size of the polymer contained in the polymer conjugateimpacts the rate of release of the agent into systemic circulation. Inone embodiment, the size of the polymer impacts the rate of release ofthe agent into systemic circulation without affecting the rate ofcleavage of the linking group. For example, with subcutaneousadministration, the rate of release of the polymer conjugate from thesubcutaneous compartment is controlled, at least in part, by the size ofthe polymer. As polymer size increases, the rate of systemic clearancefrom the subcutaneous compartment decreases. As polymer size decreases,the rate of systemic clearance from the subcutaneous compartmentincreases. As a result, the entrance of the polymer into the systemiccirculation, and subsequent cleavage of the linking group to release theagent, can be controlled.

Furthermore, the route of administration affects the rate of release ofthe agent into the systemic circulation. Administration by thesubcutaneous route results in a slower and sustained release of theagent into the systemic circulation compared to other routes ofadministration, such as for example, intravenous administration.Administration via the intravenous route results in a more rapid releaseof the agent into the systemic circulation. These concepts areillustrated in Examples 31-32 and FIGS. 2-4. Example 32 shows similarresults for pharmacokinetics in monkeys, and Example 31 shows similarresults for pharmacodynamics for rats.

The plasma concentration of rotigotine (ng/mL) after intravenous andsubcutaneous injection of POZ-rotigotine in rats is described in Example31 and shown in FIGS. 2 and 3, respectively. These results show that useof POZ conjugates of rotigotine, whether dosed intravenously (IV) orsubcutaneously (SC), will reduce the clearance rate of rotigotine fromthe blood when compared to the parent molecule alone. The terminalplasma half-life (t½) for rotigotine, POZ acetyl rotigotine and POZpropyl rotigotine was 2.8, 16 and 60 h, respectively. However, there isa difference in the PK profiles for the POZ-conjugates POZ acetylrotigotine and POZ propyl rotigotine when route of administration iscompared (IV vs SC). POZ-conjugates delivered IV are generally clearedin a bi-phasic pattern with little difference between POZ acetylrotigotine and POZ propyl rotigotine. However, when POZ acetylrotigotine and POZ propyl rotigotine are compared following SCadministration there is a marked difference. POZ acetyl rotigotine hasessentially the same PK profile when delivered either SC or IV. POZpropyl rotigotine has a markedly prolonged PK profile that is near “zeroorder” kinetics. The nature of the linker plays a role in the release ofthe agent, in this case rotigotine, and the levels measured in ratplasma from day 1 to day 7 are higher for the propyl linker than theacetyl linker. The initial plasma concentrations of rotigotine duringthe first 12 hours are lower for POZ propyl rotigotine when compared tothe POZ acetyl rotigotine conjugate. At 12 hours, the C. values ofplasma rotigotine were 6 ng/mL for POZ propyl rotigotine versus for 48ng/mL for the POZ acetyl rotigotine when dosed SC at the dose of 1.6mg/kg.

The plasma concentration of rotigotine (ng/mL) after subcutaneousinjection of POZ rotigotine in normal, treatment-naïve female macaquesmonkeys is described in Example 32 and shown in FIG. 4. Animals wererandomly assigned into four treatment groups, each N=3. Animals receivedone subcutaneous dose of either POZ alpha methyl acetyl rotigotine orPOZ propyl rotigotine at doses of either 1.5 mg/kg or 4.5 mg/kg (basedon rotigotine equivalents). The plasma concentration of rotigotine(ng/mL) after subcutaneous injection is shown in FIG. 4. These resultsshow that POZ conjugates of rotigotine will reduce the clearance rate ofrotigotine from the blood. The average terminal plasma half-life (t½) ofrotigotine from POZ alpha methyl acetyl rotigotine and POZ propionylrotigotine was 9 and 60 h, respectively. Once again, the POZ propylrotigotine has a markedly prolonged PK profile that is near “zero order”kinetics. The initial plasma concentrations of rotigotine during thefirst 12 hours are lower for POZ propyl rotigotine when compared to thePOZ alpha methyl acetyl rotigotine compound. From 4 to 192 hours, theaverage C_(ss), value of plasma rotigotine was between 1 and 6 ng/mL forPOZ propyl rotigotine at the 1.5 mg/kg dose.

These results show that controlled delivery of an agent can be “tuned”to release the agent with a desired release profile without an initialburst effect based on the nature of the releasable linker, the nature ofthe polymer, the nature of the agent, the route of administration (e.g.subcutaneous vs. IV injection) or a combination of the foregoing.

Viscosity and Drug Loading

Viscosity and drug loading are additional factors that must beconsidered when formulating a suitable polymer-drug conjugate fortreating disease. As shown in Example 30 and Table 5, higher molecularweight polymer conjugates are increasingly viscous when in solution, andthus can become too viscous for effective injection. The nature of thepolymer is also a factor in this consideration. For example, POZconjugates are less viscous than 4-arm PEG conjugates of the samemolecular weight. Similarly the PEG-dendrimer is less viscous than the4-arm PEG conjugate. Additionally, one must consider the number ofagents that can be attached to the polymer backbone. For example, thePOZ-20K polymer with 10 pendents carries more molecules of the agentthan the 4-arm PEG 20K polymer, and thus one can inject a lower mass ofPOZ conjugate and achieve the same amount of agent delivered to thesubject. Thus viscosity and drug loading, as well as the factorsaffecting release rates into the blood (discussed in above) must betaken into account when formulating a suitable polymer-drug compositionfor treating disease. In one embodiment, an acceptable polymer-drugconjugate from a viscosity standpoint is syringeabile through a 28 Gneedle. In one embodiment, an acceptable polymer-drug conjugate from aviscosity standpoint has a viscosity (as measured in mPas) of less thanor equal to 210, 175, 160, 150, 125 or 75.

Methods of Treatment

The present disclosure provides polymer conjugates comprising awater-soluble polymer and an agent, the agent linked to the polymer by areleasable linker. The present disclosure further shows that the releaseof the agent from the polymer conjugate can be controlled. In oneaspect, the agent is delivered with a pharmacokinetic profile that lackspeaks and troughs as seen in prior art treatments. In one aspect, a nearsteady state release of the agent from the polymer conjugate is achievedover a period of time from days to weeks. In one embodiment, such arelease profile provides a therapeutically effective concentration ofthe agent over such time period. As a result, the polymer conjugates ofthe present disclosure are useful for treating human disease throughappropriate selection of the agent. Furthermore, the polymer conjugatesof the present disclosure allow for less frequent administration ascompared to the art to achieve therapeutically effective concentrationsof the agent in a subject. In one embodiment, polymer conjugates of thepresent disclosure are administered once a day, once every other day,once a week or at other desired intervals.

In one embodiment, a method of treating a disease state or condition isdisclosed. Such method comprises the step of administering to thesubject an amount of a polymer conjugate of the present disclosure to asubject. In one embodiment, such disease state or condition is PD. Inone embodiment, such disease state or condition is a disease orcondition related to dopamine insufficiency in the peripheral or centralnervous system. In one embodiment, such disease or condition is restlessleg syndrome. In one embodiment, such disease state or condition is ananxiety disorder. In one embodiment, such disease state or condition isa social anxiety disorder. In one embodiment, such disease state orcondition is a panic disorder. In one embodiment, such disease state orcondition is a seizure disorder. In one embodiment, such disease stateor condition is neuropathic pain. In one embodiment, such disease stateor condition is fibromyalgia. In one embodiment, such disease state orcondition is convulsions. In one embodiment, such disease state orcondition is epilepsy. In one embodiment, such disease state orcondition is muscle tremors. In one embodiment, such disease state orcondition is muscle spasms.

In such embodiments, any polymer conjugate described herein may be usedand the agent may be selected based on the disease or condition to betreated. In a particular embodiment, the polymer is a POZ polymer. Inanother embodiment, the polymer is a PEG polymer. In still anotherembodiment, the polymer is a dextran polymer or a dextran polymermodified by oxidation.

In one embodiment, the present disclosure provides a method of treatinga disease state or condition is a disease or condition related todopamine insufficiency in the peripheral or central nervous system. Suchmethod comprises the step of administering to the subject an amount of apolymer conjugate of the present disclosure to a subject.

In one embodiment, the disease or condition related to dopamineinsufficiency is PD. Therefore, the present disclosure provides a methodof treating PD. Such method comprises the step of administering to thesubject an amount of a polymer conjugate of the present disclosure to asubject.

In one embodiment, the disease or condition related to dopamineinsufficiency is restless leg syndrome. Therefore, the presentdisclosure provides a method of treating restless leg syndrome. Suchmethod comprises the step of administering to the subject an amount of apolymer conjugate of the present disclosure to a subject.

Any polymer conjugate of the present disclosure may be used in themethods above. In a particular embodiment, the following polymerconjugates may be used in such methods of treatment.

In one embodiment, the polymer conjugate is a poly(oxazoline) polymerconjugate and the agent is a compound useful in the treatment of PD oranother disease or condition related to dopamine insufficiency in theperipheral or central nervous system.

In one embodiment, the polymer conjugate is a poly(oxazoline) polymerconjugate and the agent is a dopamine agonist, adenosine A_(2A)antagonist, anticholinergic, monamine oxidase-B inhibitor orcatechol-O-methyl transferase (COMT) inhibitor.

In one embodiment, the polymer conjugate is a poly(oxazoline) polymerconjugate and the agent is rotigotine, pramipexole, quinagolide,fenoldopam, apomorphine, 5-OH-DPAT, ropinirole, pergolide, cabergoline,or bromocriptine.

In one embodiment, the polymer conjugate is a poly(oxazoline) polymerconjugate and the agent is rotigotine or (-)rotigotine.

In one embodiment, the polymer conjugate is a poly(oxazoline) polymerconjugate and the agent is ropinirole.

In one embodiment, the polymer conjugate is a poly(oxazoline) polymerconjugate and the agent is trihexyphenidyl, biperidin or hyoscyamine.

In one embodiment, the polymer conjugate is a poly(oxazoline) polymerconjugate and the agent is seligiline or rasagiline.

In one embodiment, the polymer conjugate is a poly(oxazoline) polymerconjugate and the agent is tolcapone or entacapone.

In one embodiment, the polymer conjugate is a poly(oxazoline) polymerconjugate and the agent is caffeine, theophylline, istradefylline orpreladenant.

In the foregoing embodiments where the polymer conjugate is apoly(oxazoline) polymer conjugate, the poly(oxazoline) polymer conjugatemay have the general formula as shown for compound II, IIA, IIB or IIC.In one embodiment, the polymer conjugate is a poly(oxazoline) polymerconjugate having the general formula as shown for compound IIC or anexample herein.

In one embodiment, the polymer conjugate is a polyethylene glycolpolymer conjugate and the agent is a compound useful in the treatment ofPD or another disease or condition related to dopamine insufficiency inthe peripheral or central nervous system.

In one embodiment, the polymer conjugate is a polyethylene glycolpolymer conjugate and the agent is a dopamine agonist, adenosine A_(2A)antagonist, anticholinergic, monamine oxidase-B inhibitor orcatechol-O-methyl transferase (COMT) inhibitor.

In one embodiment, the polymer conjugate is a polyethylene glycolpolymer conjugate and the agent is rotigotine, pramipexole, quinagolide,fenoldopam, apomorphine, 5-OH-DPAT, ropinirole, pergolide, cabergoline,or bromocriptine.

In one embodiment, the polymer conjugate is a polyethylene glycolpolymer conjugate and the agent is rotigotine or (-)rotigotine.

In one embodiment, the polymer conjugate is a polyethylene glycolpolymer conjugate and the agent is ropinirole.

In one embodiment, the polymer conjugate is a polyethylene glycolpolymer conjugate and the agent is trihexyphenidyl, biperidin orhyoscyamine.

In one embodiment, the polymer conjugate is a polyethylene glycolpolymer conjugate and the agent is seligiline or rasagiline.

In one embodiment, the polymer conjugate is a polyethylene glycolpolymer conjugate and the agent is tolcapone or entacapone.

In one embodiment, the polymer conjugate is a polyethylene glycolpolymer conjugate and the agent is caffeine, theophylline,istradefylline or preladenant.

In the foregoing embodiments, when the polymer is a polyethylene glycolpolymer, the polyethylene glycol polymer may be a multi-arm polymer,including a 4-arm polymer, a difuncitonal polymer or a dendrimer.

In the foregoing embodiments where the polymer conjugate is apolyethylene glycol polymer conjugate, the polyethylene glycol polymerconjugate may have the general formula as shown for compound I or anexample herein.

In one embodiment, the polymer conjugate is a dextran or oxidizeddextran polymer conjugate and the agent is a compound useful in thetreatment of PD or another disease or condition related to dopamineinsufficiency in the peripheral or central nervous system.

In one embodiment, the polymer conjugate is a dextran or oxidizeddextran polymer conjugate and the agent is a dopamine agonist, adenosineA_(2A) antagonist, anticholinergic, monamine oxidase-B inhibitor orcatechol-O-methyl transferase (COMT) inhibitor.

In one embodiment, the polymer conjugate is a dextran or oxidizeddextran polymer conjugate and the agent is rotigotine, pramipexole,quinagolide, fenoldopam, apomorphine, 5-OH-DPAT, ropinirole, pergolide,cabergoline, or bromocriptine.

In one embodiment, the polymer conjugate is a dextran or oxidizeddextran polymer conjugate and the agent is rotigotine or (-)rotigotine.

In one embodiment, the polymer conjugate is a dextran or oxidizeddextran polymer conjugate and the agent is ropinirole.

In one embodiment, the polymer conjugate is a dextran or oxidizeddextran polymer conjugate and the agent is trihexyphenidyl, biperidin orhyoscyamine.

In one embodiment, the polymer conjugate is a dextran or oxidizeddextran polymer conjugate and the agent is seligiline or rasagiline.

In one embodiment, the polymer conjugate is a dextran or oxidizeddextran polymer conjugate and the agent is tolcapone or entacapone.

In one embodiment, the polymer conjugate is a dextran or oxidizeddextran polymer conjugate and the agent is caffeine, theophylline,istradefylline or preladenant.

In the foregoing embodiments where the polymer conjugate is a dextran oroxidized dextran polymer conjugate, the dextran or oxidized dextranpolymer conjugate may have the general formula as shown for compound Ior an example herein.

In one embodiment, the present disclosure provides a method of treatinga disease or condition caused by excessive GABA re-uptake or GABAre-uptake. In another embodiment, the present disclosure provides amethod of treating an anxiety disorder, social anxiety disorder, panicdisorder, neuropathic pain (which includes usefulness in poorlyunderstood disorders like fibromyalgia), chronic pain, muscle tremors,muscle spasms, seizures, convulsions and/or epilepsy. Such methodcomprises the step of administering to the subject an amount of apolymer conjugate of the present disclosure to a subject. In such anembodiment, the agent may be a GABA re-uptake inhibitor.

In one embodiment, the disease or condition caused by excessive GABAre-uptake or GABA re-uptake is an anxiety disorder. Therefore, thepresent disclosure provides a method of treating an anxiety disorder.Such method comprises the step of administering to the subject an amountof a polymer conjugate of the present disclosure to a subject.

In one embodiment, the disease or condition caused by excessive GABAre-uptake or GABA re-uptake is a social anxiety disorder. Therefore, thepresent disclosure provides a method of treating a social anxietydisorder. Such method comprises the step of administering to the subjectan amount of a polymer conjugate of the present disclosure to a subject.

In one embodiment, the disease or condition caused by excessive GABAre-uptake or GABA re-uptake is a panic disorder. Therefore, the presentdisclosure provides a method of treating a panic disorder. Such methodcomprises the step of administering to the subject an amount of apolymer conjugate of the present disclosure to a subject.

In one embodiment, the disease or condition caused by excessive GABAre-uptake or GABA re-uptake is a seizure disorder. Therefore, thepresent disclosure provides a method of treating a seizure disorder.Such method comprises the step of administering to the subject an amountof a polymer conjugate of the present disclosure to a subject.

In one embodiment, the disease or condition caused by excessive GABAre-uptake or GABA re-uptake is muscle tremors. Therefore, the presentdisclosure provides a method of treating muscle tremors. Such methodcomprises the step of administering to the subject an amount of apolymer conjugate of the present disclosure to a subject.

In one embodiment, the disease or condition caused by excessive GABAre-uptake or GABA re-uptake is muscle spasms. Therefore, the presentdisclosure provides a method of treating muscle spasms. Such methodcomprises the step of administering to the subject an amount of apolymer conjugate of the present disclosure to a subject.

In one embodiment, the disease or condition caused by excessive GABAre-uptake or GABA re-uptake is convulsions. Therefore, the presentdisclosure provides a method of treating convulsions. Such methodcomprises the step of administering to the subject an amount of apolymer conjugate of the present disclosure to a subject.

In one embodiment, the disease or condition caused by excessive GABAre-uptake or GABA re-uptake is neuropathic pain. Therefore, the presentdisclosure provides a method of treating neuropathic pain. Such methodcomprises the step of administering to the subject an amount of apolymer conjugate of the present disclosure to a subject.

In one embodiment, the disease or condition caused by excessive -GABAre-uptake or GABA re-uptake is fibromyalgia. Therefore, the presentdisclosure provides a method of treating fibromyalgia. Such methodcomprises the step of administering to the subject an amount of apolymer conjugate of the present disclosure to a subject.

In one embodiment, the disease or condition caused by excessive. GABAre-uptake or GABA re-uptake is epilepsy. Therefore, the presentdisclosure provides a method of treating epilepsy. Such method comprisesthe step of administering to the subject an amount of a polymerconjugate of the present disclosure to a subject.

In one embodiment, the disease or condition caused by excessive GABAre-uptake or GABA re-uptake is muscle spasms. Therefore, the presentdisclosure provides a method of treating muscle spasms. Such methodcomprises the step of administering to the subject an amount of apolymer conjugate of the present disclosure to a subject.

In one embodiment, the disease or condition caused by excessive GABAre-uptake or GABA re-uptake is insomnia. Therefore, the presentdisclosure provides a method of treating insomnia. Such method comprisesthe step of administering to the subject an amount of a polymerconjugate of the present disclosure to a subject.

Any polymer conjugate of the present disclosure may be used in themethods above. In a particular embodiment, the following polymerconjugates may be used in such methods of treatment.

In one embodiment, the polymer conjugate is a poly(oxazoline) polymerconjugate and the agent is a compound useful in the treatment of ananxiety disorder, social anxiety disorder, panic disorder, neuropathicpain (which includes usefulness in poorly understood disorders likefibromyalgia), chronic pain, muscle tremors, muscle spasms, seizures,convulsions and/or epilepsy.

In one embodiment, the polymer conjugate is a poly(oxazoline) polymerconjugate and the agent is a GABA re-uptake inhibitor.

In one embodiment, the polymer conjugate is a poly(oxazoline) polymerconjugate and the agent is tiagabine or nipecotic acid.

In one embodiment, the polymer conjugate is a poly(oxazoline) polymerconjugate and the agent is tiagabine.

In the foregoing embodiments where the polymer conjugate is apoly(oxazoline) polymer conjugate, the poly(oxazoline) polymer conjugatemay have the general formula as shown for compound II, IIA, IIB or IIC.In one embodiment, the polymer conjugate is a poly(oxazoline) polymerconjugate having the general formula as shown for compound IIC or anexample herein.

In one embodiment, the polymer conjugate is a polyethylene glycolpolymer conjugate and the agent is a compound useful in the treatment ofan anxiety disorder, social anxiety disorder, panic disorder,neuropathic pain (which includes usefulness in poorly understooddisorders like fibromyalgia), chronic pain, muscle tremors, musclespasms, seizures, convulsions and/or epilepsy.

In one embodiment, the polymer conjugate is a polyethylene glycolpolymer conjugate and the agent is a GABA re-uptake inhibitor.

In one embodiment, the polymer conjugate is a polyethylene glycolpolymer conjugate and the agent is tiagabine or nipecotic acid.

In one embodiment, the polymer conjugate is a polyethylene glycolpolymer conjugate and the agent is tiagabine.

In the foregoing embodiments, when the polymer is a polyethylene glycolpolymer, the polyethylene glycol polymer may be a multi-arm polymer,including a 4-arm polymer, a difuncitonal polymer or a dendrimer.

In the foregoing embodiments where the polymer conjugate is apolyethylene glycol polymer conjugate, the polyethylene glycol polymerconjugate may have the general formula as shown for compound I or anexample herein.

In one embodiment, the polymer conjugate is a dextran or oxidizeddextran polymer conjugate and the agent is a compound useful in thetreatment of an anxiety disorder, social anxiety disorder, panicdisorder, neuropathic pain (which includes usefulness in poorlyunderstood disorders like fibromyalgia), chronic pain, muscle tremors,muscle spasms, seizures, convulsions and/or epilepsy.

In one embodiment, the polymer conjugate is a dextran or oxidizeddextran polymer conjugate and the agent is a GABA re-uptake inhibitor.

In one embodiment, the polymer conjugate is a dextran or oxidizeddextran polymer conjugate and the agent is tiagabine or nipecotic acid.

In one embodiment, the polymer conjugate is a dextran or oxidizeddextran polymer conjugate and the agent is tiagabine.

In the foregoing embodiments where the polymer conjugate is a dextran oroxidized dextran polymer conjugate, the dextran or oxidized dextranpolymer conjugate may have the general formula as shown for compound Ior an example herein.

In the methods described, the polymer conjugate may be administeredalone or as a part of a pharmaceutical composition as described herein.In one embodiment, the subject is determined to be in need of suchtreatment. In a further embodiment, the polymer conjugate isadministered in a therapeutically effective amount. In the methodsdisclosed herein, the subject may be a mammal. In certain embodiments,the subject is a human.

In one embodiment, the methods of treatment are accomplished bysubcutaneous administration of the polymer conjugates of the presentdisclosure or pharmaceutical compositions containing such polymerconjugates.

In addition, in one embodiment, such polymer conjugate is administeredonce a day. In another embodiment, such polymer conjugate isadministered once every other day. In still a further embodiment, suchpolymer conjugate is administered every third day, every fourth day,every fifth day or every sixth day. In yet a further embodiment, suchpolymer conjugate is administered once a week. Other dosing frequenciesmay also be used based on the nature of the polymer conjugate selectedand the release kinetics of the agent.

The polymer conjugates described herein can also be administered incombination with other therapeutic agents, for example, other agentsthat are useful for treatment of PD or any other condition recitedherein. When administered with other therapeutic agents, the polymerconjugates of the present disclosure may be administered before, afteror at the same time as the additional therapeutic agent. Accordingly, inone embodiment the present disclosure also provides a compositioncomprising a polymer conjugate described herein, at least one othertherapeutic agent, and a pharmaceutically acceptable diluent or carrier.

Kits

The present disclosure provides a kit comprising, consisting essentiallyof or consisting of a polymer conjugate of the present disclosure,packaging material, and instructions for administering the foregoing toa subject for the treatment of PD or another disease or conditionrelated to dopamine insufficiency in the peripheral or central nervoussystem.

The present disclosure also provides a kit comprising, consistingessentially of or consisting of a polymer conjugate of the presentdisclosure, packaging material, and instructions for administering theforegoing to a subject for the treatment of an anxiety disorder, socialanxiety disorder, panic disorder, neuropathic pain (which includesusefulness in poorly understood disorders like fibromyalgia), chronicpain, muscle tremors, muscle spasms, seizures, convulsions and/orepilepsy.

The present disclosure provides a kit comprising, consisting essentiallyof or consisting of a polymer conjugate of the present disclosure, atleast one other therapeutic agent, packaging material, and instructionsfor administering the foregoing to a subject for the treatment of PD oranother disease or condition related to dopamine insufficiency in theperipheral or central nervous system.

The present disclosure also provides a kit comprising, consistingessentially of or consisting of a polymer conjugate of the presentdisclosure, at least one other therapeutic agent, packaging material,and instructions for administering the foregoing to a subject for thetreatment of an anxiety disorder, social anxiety disorder, panicdisorder, neuropathic pain (which includes usefulness in poorlyunderstood disorders like fibromyalgia), chronic pain, muscle tremors,muscle spasms, seizures, convulsions and/or epilepsy.

Methods of Manufacture

In one embodiment, the agent is linked to the polymer using “clickchemistry”. This approach is also readily applicable to all polymertypes. In one embodiment, the polymer is POZ. In another embodiment, thepolymer is PEG. In another embodiment, the polymer is dextran. The clickchemistry approach involves the reaction between an alkyne group and anazido group. Therefore, in one embodiment, the agent contains one of analkyne or azido group and the polymer contains the other of the alkyneor azido group. The respective groups may also be present on linkinggroups attached to the agent and/or polymer as well. In one aspect, theclick chemistry reaction involves the reaction of an azidoester on theagent and an alkyne on the polymer. In a particular embodiment of thisaspect, the azidoester group is formed by suitable chemical reactionswith a chemical group on the agent, such as, but not limited to, ahydroxyl group. An exemplary reaction would be the preparation of anazidoester by displacing a halide from a halo acid with sodium azide toform the azidoacid followed by esterification of the azidoacid with ahydroxyl group on the agent (exemplified here as rotigotine).

The azidorotigotine ester is then linked to an alkyne functionalitypresent on the polymer. In a particular embodiment, the alkynefunctionality is an acetylene functionality present at a pendentposition on the POZ polymer.

While the above method may be used, other approaches to the formation ofreleasable functionalities may be used. For example, a linkagecontaining an ester as the cleavable moiety may also be formed bycreating an azide functional group on the polymer, such as a pendentgroup on a POZ polymer, creating an alkyne group on the agent, such asan acetylene ester of rotigotine, and reacting the azide group and thealkyne group to form a linkage having a cleavable moiety (in this casean ester bond).

In another approach, a carboxylic acid group can be created on thepolymer, such as a pendent group on a POZ polymer, and reacting thecarboxylic acid group by directly esterifying an alcohol or phenolicgroup on the agent to form a linkage having a cleavable moiety (in thiscase an ester bond). In one embodiment, a carboxylic acid group on thePOZ polymer is generated at a pendent position on the POZ polymer byincluding a carboxylated monomer in the polymerization reaction.

In the preparation of the polymer conjugates of the present disclosure,the number of agents on the polymer is controlled by the number ofreactive groups present on the polymer; in one embodiment, the reactivegroups are present in a pendent position on the polymer. For reactivegroups at the pendent position, the number of reactive groups present onthe polymer is controlled by the ratio of monomer units (for example,monomer oxazolines) having functionalized side chains (e.g.. acetylenes)capable of forming linkages with the agent or linking group relative tomonomer units having inactive side-chains (e.g. alkyls) used in thepolymerization. In addition, for a given ratio of monomer units havingfunctionalized side chains, the polymer length can be controlledproviding further control of the number of agents loaded onto a givenpolymer conjugate. Therefore, the number of agents attached to aparticular polymer conjugate can be controlled. As described above, thenature of the linking group, the size of the polymer and the route ofadministration (intravenous, subcutaneous or transdermal) allows controlover the release kinetics of the agent from the polymer. These combinedproperties allow one to “tune” the release of the attached agent byvarying the amount of agent delivered and varying the release kineticsof the agent for the desired pharmacology.

Pharmaceutical Compositions

Polymer conjugates can be formulated for both human and veterinary use.These formulations contain pharmaceutically accepted ingredients thatact as fillers, binders, carriers, stabilizers, buffers, solvents,co-solvents, viscosity enhancers, lubricants, surfactants, flavoring andsweetening agents, taste-masking agents, inorganic salts, antioxidants,antimicrobial agents, chelating agents, lipids, phospholipids, (Ref:Handbook of Pharmaceutical Excipients, 3rd edition, Ed. A. H. Kibbe,Pharmaceutical Press, 2000). The amount of agent in these formulationswill depend on their physicochemical properties, dose and mode ofadministration. Most dosage forms will generally contain 1 to 99% byweight of the total formulation.

Formulations suitable for oral administration can be in solid form andthey include tablets, pills, capsules, cachets, lozenges, fastdissolving solids, fine powders and granular powders. A tablet is acompression or mold of the drug conjugate and acceptable pharmaceuticalexcipients. Capsules are gelatin and non-gelatin cachets thatencapsulate the drug and excipients. Formulations are also in liquidform and they include solutions, suspensions, emulsions, syrups andelixirs. These liquids may be aqueous, sugar based and non-aqueousbased, glycol based.

Formulations suitable for parenteral use are sterile liquids and sterilepowders and lyophilized powders ready for reconstitution in a suitableaqueous medium. Examples of the latter are sterile water for injection,5% dextrose solution for injection, and 0.9% sodium chloride solutionfor injection, and lactated Ringer's injection. These formulations canbe administered intravenously, subcutaneously, intramuscularly, andintradermally. These formulations are pH balanced and isotonic to bloodand surrounding tissue. Similar formulations can be delivered as nasalsprays and eye drops.

Topical, transdermal and rectal formulations are water, polymer and oilbased. They can be dissolved or suspended in mineral oil, petroleumwaxes, liquid and solid polyols, polyhydroxy alcohols, cocoa butter,hydrogenated fats, surfactants, and esters of carboxylic acids.Transdermal formulations are reservoir or monolithic in design and thedrug conjugates are typically in soluble form. Transdermal formulationsalso contain excipients to promote permeation of the agent across theskin.

EXPERIMENTAL EXAMPLES Example 1—Synthesis of randomH-[(Ptyn)₁₀(EOZ)190]-T-CO₂H

The synthesis of POZ polymers with various pendent groups is describedin U.S. Pat. Nos. 8,110,651 and 8,101,706, each of which is incorporatedherein by reference for such teachings. In a specific embodiment, thesynthesis of H-[(Ptyn)₁₀(EOZ)₁₉₀]-T-CO₂H is provided although other POZpolymers with different molecular weights, different initiating andterminating groups as well as different groups at the “R₂” position(with reference to the definitions of POZ above) may be produced by thesame methods. In addition, block copolymers may be produced in additionto the random copolymers described below. Methods for producing randomand block copolymers are described in U.S. patent application Ser. Nos.12/744,472 and 12/787,241, each of which is incorporated herein byreference for such teachings.

For the synthesis of H-[(Ptyn)₁₀(EOZ)₁₉₀]-T-CO₂H, triflic acid (HOTf,173.3 μL, 1.96 mmol) was added to a solution of 2-pentynyl-2-oxazoline(PtynOZ, 3.76 g, 27.4 mmol, 14 eq) and 2-ethyl-2-oxazoline (EOZ, 46.61g, 470.2 mmol, 240 eq) in chlorobenzene (124 mL). After stirring for 5minutes at room temperature, the mixture was heated to 80° C. for 10hours followed by cooling to room temperature. In a separate flask, theterminating reagent was prepared by the dropwise addition of methyl3-mercaptopropionate (1.23 mL, 0.0114 mol) into a suspension of sodiumhydride (60% in mineral oil, 0.272 g, 0.0068 mol) in chlorobenzene (34mL). This mixture was stirred for 7 hours, before the solution of livingpolymer of H-(Ptyn)10(EOZ)200⁺ was added. The resulting mixture was thenstirred for 18 hours. The solvent was removed by rotary evaporation toyield a white residue. This residue was dissolved in water and the pHadjusted to 12.0. The resulting aqueous solution was purified byion-exchange chromatography using DEAE Sepharose FF. The aqueoussolution was saturated with NaCl (15% w/w) and extracted withdichloromethane. The combined organic phases were dried over anhydroussodium sulfate, filtered, and concentrated using a rotary evaporator.The residue was precipitated by adding the dichloromethane concentrateto diethyl ether. The precipitated material was collected and dried invacuo to give 22.8 g of desired product as a white powder (50% yield).

¹ H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed the usual backbonepeaks at 1.13 ppm (m, 3H, CH₃CH₂CO—); 2.32 ppm (m) and 2.41 (s) (totalarea 2H, CH₃CH₂CO—); and 3.47 ppm (m, 4H, —NCH₂CH₂N—). The terminalgroup peaks appear at 2.63 ppm (m, 2H, —SCH₂CH₂CO₂H), 2.74 ppm (m, 2H,—CH₂SCH₂CH₂ CO₂H), and 2.85 ppm (m, 2H, —SCH₂CH₂CO₂H). The pendentpentynyl group peaks appear at 1.85 ppm (m, 2H, —CH₂CH₂CCH) and 2.03 ppm(br s, 1H, —CH₂CH₂C_CH). The number of pendent, Ptyn, groups weredetermined as 8.5 by comparing the integrations of terminal acetyleneproton and polymer backbone protons. GPC gave Mn=19,500 Da and Mp=20,800Da with PDI of 1.07.

Example 2—Synthesis of Azidoacetic Acid in Non-aqueous Solvents

This example provides a general synthetic scheme for the synthesis ofvarious azidoalkyl acid linkers. To exemplify this method, the synthesisof 2-azidoacetic acid is provided. Through the substitution of2-bromoacetic acid, used in the synthesis of 2-azidoacetic acid, withother reagents azidoalkyl acid linkers, such as, but not limited to,3-azidopropionic acid and 2-azoidopropioni acid, may be produced.

For the synthesis of 2-azidoacetic acid, to a solution of 2-bromoaceticacid (1 g, 7.20 mmol) in DMF (14.39 ml) was added sodium azide (0.491 g,7.56 mmol). After stirring for 16 hours at room temperature, thereaction mixture was monitored by RP HPLC showing 98% conversion(retention time, t_(r)=2.40 min) with remaining 2% bromoacetic acid(t_(r)=2.77 min).

H¹ NMR analysis (10 mg/mL in CDCl₃) showed the relevant peak at 3.84 ppm(s, 2H, N₃CH₂CO₂H).

Example 3—Synthesis of Rotigotine with 2-azidoacetic Acid Linker

In a 25 mL round bottom flask, was placed rotigotine (1 g, 3.17 mmol, 1equiv.), 2-azidoacetic acid-DMAP salt (0.849 g, 3.80 mmol, 1.2 equiv.)and 32 mL of anhydrous DCM and the mixture stirred under argon. DMAP(0.077 g, 0.634 mmol, 0.2 equiv.) and DCC (0.785 g, 3.80 mmol, 1.2equiv.) were added as solids. The mixture was stirred for 16 hours atroom temperature. The mixture was then filtered to remove precipitatedurea and concentrated using a rotary evaporator. The crude mixture wasfirst purified by silica gel column chromatography using a mixture ofethyl acetate and hexanes (1:2) as an eluent to give a clear yellow oil(1.27 g, 92% yield).

A second purification was performed by reversed phase chromatography toremove free rotigotine and other small molecule impurities. A samplesolution for loading was prepared by dissolving crudeazidoacetyl-rotigotine (350 mg) in 0.1% TFA in acetonitrile (4.05 mL),followed by addition of 1 N HCl (0.91 mL) and 0.1% TFA in water (4.04mL). The sample solution was filtered through a 0.2 um PTFE syringefilter, and then was loaded to a Waters SunFire Prep C18 OBD 30/250Column (from Waters) on an AKTA Purifier system equipped with an UVdetector at 214 nm. 0.1% TFA in water (A) and 0.1% TFA in acetonitrile(B) were used as mobile phase. The column was then eluted isocraticallywith 40% of mobile phase B at flow rate of 20 mL/min. The fractions thatcontained azidoacetyl-rotigotine were collected and pooled. Acetonitrilein the pooled fraction was evaporated by rotary-evaporation. Theremaining aqueous solution was extracted with DCM (3×50 mL), dried overanhydrous sodium sulfate and filtered, followed by evaporation of theDCM. The residue was dried in vacuum (293 mg, 83%).

¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed peaks at 0.90 ppm (t,J=6.84 Hz, 3H), 1.25 (in, 1H), 1.29 (m, 1H), 1.49 (m, 1H), 1.59 (m, 1H),2.05 (m, 2H), 2.54 (m, 3H), 2.82 (m, 3H), 2.97 (m, 3H), 4.156 N₃CH₂ C(═O)O— (s,2H), 6.81 (s, 1H), 6.88 (d, J=7.81 Hz, 1H), 6.92 (t, J=3.42 Hz,1H), 7.02 (d, J=7.32 Hz, 1H), 7.13 (m, 2H).

RP-HPLC analysis showed that the product contained no free rotigotine.The HPLC chromatogram of azidoacetyl-rotigotine before (FIG. 1A) andafter (FIG. 1B) reversed phase chromatography purification are shown.

Example 4—Synthesis of Rotigotine with 3-azidopropionic Acid Linker

In a 50 mL round bottom flask, rotigotine (500 mg, 1.56 mmol, 1 equiv.),3-azidopropionic acid (447 mg, 3.73 mmol, 2.4 equiv.) dissolved in 5 mLDCM, pyridine (302 μL, 3.73 mmol, 2.4 equiv.) were dissolved in 50 mLanhydrous DCM and allowed to stir under argon. The solution was cooledin an ice-water bath for 5 min, and the bath was removed. To thesolution DCC was added (778 mg, 3.73 mmol, 2.4 equiv.). The solution wasallowed to stir at room temperature under argon. Following an overnightreaction, reverse phase HPLC analysis of the reaction mixture showedcomplete conversion of free rotigotine to the ester form. The reactionmixture was filtered and the filtrate was concentrated to dryness on arotary-evaporator. The crude product was then purified by silica gelchromatography. The crude product was dissolved in a mixed solvent ofhexanes-ethyl acetate (6 mL, 4:1 v/v), was then loaded onto a 300 mLSilica Gel Column (30 mm id). The column was eluted with hexanes-ethylacetate mixed solvent (4:1 v/v). The fractions (10 mL each) wereanalyzed by TLC and reversed phase HPLC. The product fractions werepooled, evaporated by rotary-evaporation, and then dried under vacuumovernight. Yield: 292 mg.

¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed peaks at 3.706 ppmN₃CH₂CH₂C(═O)O— (t, 2H), 2.838 N₃ CH₂CH₂C(═O)O— (t, 2H).

Example 5—Synthesis of Rotigotine with 2-azidopropionic Acid Linker

In a 100 mL round bottom flask was placed 2-azidopropionic acid (251 mg,2.02 mmol, 1.3 equiv.) dissolved in 3 mL of DCM, rotigotine (500 mg,1.55 mmol, 1 equiv.), and 4-DMAP (249 mg, 2.02 mmol, 1.3 equiv.)dissolved in 6 mL of DCM (6 mL) and the mixture was allowed to stirunder argon. The solution was cooled by placing the flask in anice-water bath for 5min. To the solution, DCC was added (421 mg, 2.02mmol, 1.3 equiv.). The progress of the reaction was followed by reversedphase HPLC. Following overnight stirring at room temperature, additional2-azidopropionic acid (126 mg, 0.65 equiv.) in 2 mL of DCM and 4-DMAP(124 mg, 0.65 equiv.) were added to the reaction mixture, followed byDCC (211 mg, 0.65 equiv.). The solution was allowed to stir at roomtemperature for another 3.5 hours. HPLC result shows 94% of conversionto ester. The reaction mixture was filtered and the filtrate wasconcentrated to dryness on a rotary-evaporator. The crude product wasthen purified by silica gel chromatography. The crude product wasdissolved in a mixed solvent of hexanes-ethyl acetate (6 mL, 4:1 v/v),and then loaded on to a 300 mL Silica Gel Column (30 mm id). The columnwas eluted with a hexanes-ethyl acetate mixed solvent (4:1 v/v). Thefractions (10 mL each) were analyzed by TLC and reversed phase HPLC. Theproduct fractions were pooled, evaporated by rotary-evaporation, andthen dried in vacuum overnight. Yield: 307 mg.

Example 6—Preparation of H-[(Acetyl-Rotigotine)₁₀(EOZ)₁₉₀]-COOH 20K byAttachment of Azidoacetyl-Rotigotine to Polyoxazoline 10 Pendent Acid20K

H-[(PtynOZ)10(EOZ)1901]-COOH 20K polymer (1.306 gm, 0.0653 mmol, 1.0equiv.; prepare as described in Example 1) was dissolved in 15 mL of THFin a 100 mL round bottom flask. In a separate 50 mL round bottom flask,azidoacetyl-rotigotine (FW 384.50 Da. 251 mg, 0.653 mmol, 10.0 equiv.;prepared as in Example 3) was dissolved in 15 mL of THF (15 mL). Theazidoacetyl-rotigotine solution was transferred into the 100 mL roundbottom flask. The solution was flushed with argon. CuI (Copper (I)iodide, ≧99.5%, Sigma-Aldrich, 50 mg, 0.261 mmol, 4.0 equiv.) was thenadded to the flask, followed by addition of TEA (127 μL, 0.914 mmol,14.0 equiv.). The solution was allowed to stir overnight at 45° C. underArgon. The green, crude reaction mixture was filtered with the aid of a0.2 μm syringe filter, and then 0.1 N HCl acid (20 mL) was added intothe filtrate. The mixture turned brown in color. The THF in the mixturewas evaporated with the aid of a rotary-evaporator at 28° C.

Two column purification steps were employed to purify the crude product.In step one, a glass column (2 cm ID) was packed with a slurry of silicagel 60 (EMD, 70-230 Mesh, 30 mL) in 60 mL of 0.1 N HCl acid. The columnpacking and elution was done by gravity. Prewashed (water and 2 mM HClacid) Dowex® M4195 media (20 mL) was packed above the silica layer. Thecolumn was equilibrated with 2 mM HCl (50 mL).

In a second glass column, Amberlite IR-120H (40 mL) was packed andwashed with deionized water until the conductivity of the eluent wasless than 1 μS/cm. The column was then equilibrated with 2 mM HCl (40mL).

The filtered crude reaction mixture (20 mL) which contained >300 mg/LCu^(+/2+) (measured by Quantofi Copper test stick), was loaded on to thefirst Dowex/silica gel column. The column was eluted with 2 mM HCl acid.The eluent that containing the H-[(Acetyl-Rotigotine)₁₀(EOZ)₁₉₀]—COOH20K polymer product (100 mL) was collected. The Cu^(+/2+) levels wasless than 10 mg/L (Quantofi Copper test stick). Free rotigotine in theeluent was then removed by the Amberlite IR-120H as next described. Theeluent of the Dowex/silica gel column (100 mL) was loaded onto AmberliteIR-120H (40 mL) column. The column was eluted with 1 mM HCl. To theeluent (150 mL) from the Amberlite column, NaCl was added to make 10%concentration. The cloudy solution was extracted with DCM (3×200 mL,gentle shaking) and dried over anhydrous sodium sulfate. The salt wasfiltered off, and the filtrate was concentrated to ˜20 mL byrotary-evaporation. The concentrated solution was added to 400 mL ofethyl ether to obtain a precipitate. Following filtration, theprecipitate was dried under vacuum. The yield was 1.13 gm. RP-HPLCanalysis showed the absence of rotigotine and azidoacetyl-rotigotine.The produced polyoxazoline conjugate of rotigotine showed good watersolubility.

¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed peaks at 5.479 ppm —NCH₂C(═O)O— (s,2H), 6.945-7.197 from the phenyl and thiophene groups ofrotigotine.

Example 7—Preparation of H-[(Propionyl-Rotigotine)₁₀(EOZ)₁₉₀]-COOH 20Kby attachment of 3-Azidopropionyl-Rotigotine to Polyoxazoline 10 PendentAcid 20K

H-[(PtynOZ)₁₀(EOZ)190]-COOH 20K (681 mg, 0.034 m mol, 1 equiv.; preparedas in Example 1) was dissolved in 15 mL of THF in a 50 mL round bottomflask. In a 20 mL glass vial, 3-azidopropionyl-rotigotine (140 mg, 0.340mmol, 10.0 equiv.; prepared as in Example 4) was dissolved in 5 mL ofTHF. The 3-azidopropionyl-rotigotine solution was transferred into the50 mL round bottom flask. The solution was flushed under Argon. CuI(Copper (I) iodide, ≧99.5%, Sigma-Aldrich, 26 mg, 0.136 mmol, 4.2equiv.) was then added to the flask, followed by addition of TEA (20 μL,0.144 mmol). The solution was allowed to stir overnight at 45° C. underan Argon atmosphere. The green crude reaction mixture was cooled to roomtemperature and 0.1 N HCl acid (10 mL) was added to it. The reactionmixture became a clear yellow-brownish color. The THF in the mixture wasevaporated with the aid of a rotary-evaporator at 28° C.

The reaction mixture was purified, extracted and precipitated asexplained in Example 6. The yield was 611 mg. RP-HPLC analysis showedthe absence of rotigotine and 3-azidopropionyl-rotigotine. The producedpolyoxazoline conjugate of rotigotine showed good water solubility.

¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed peaks at 4.829 ppm —NCH₂CH₂C(═O)O— (t, 2H), 6.876-7.194 from the phenyl and thiophene groups ofrotigotine.

Example 8—Preparation ofH-[(-[(α-Methyl-Acetyl-Rotigotine)₁₀(EOZ)₁₉₀]-COOH 20K by Attachment of2-Azidopropionyl-Rotigotine to Polyoxazoline 10 Pendent Acid 20K

H-[(PtynOZ)₁₀(EOZ)₁₉₀]COOH 20K (1.409 gm, 0.070 mmol, 1 equiv.; preparedas in Example 1) was dissolved in 15 mL of in a 100 mL round bottomflask. In a 20 mL glass vial, 2-azidopropionyl-rotigotine (291 mg, 0.705mmol, 10.0 equiv.; prepared as in Example 5) was dissolved in 15 mL ofTHF (15 mL). The 2-azidopropionyl-rotigotine solution was transferredinto the 100 mL round bottom flask. The solution was flushed underargon. CuI (Copper (I) iodide, ≧99.5%, Sigma-Aldrich, 54 mg, 0.282 mmol,4.0 equiv) was then added to the flask, followed by addition of TEA (41μL, 0.296 mmol, 4.2 equiv.). The solution was stirred overnight at 45°C. under an argon atmosphere. The reaction mixture was cooled to roomtemperature, filtered through a 0.2 um PTFE syringe filter. 0.1 N HCl(20 mL) and added into the filtrate. The crude mixture turned clearbrown in appearance. The THF in the mixture was evaporated with the aidof a rotary-evaporator at 28° C.

The reaction mixture was purified, extracted and precipitated asdescribed in Example 6. The yield was 541 mg. RP-HPLC analysis showedthe absence of rotigotine and 2-azidopropionyl-rotigotine. The producedpolyoxazoline conjugate of rotigotine showed good water solubility.

¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed peaks at 5.692 ppm—N(CH₂)CHC(═O)O— (s, H), 6.943-7.196 from the phenyl and thiophenegroups of rotigotine.

Example 9—Preparation of 4-Arm Polyethylene Glycol-acetylene (10K)

4-Arm Polyethylene Glycol-SCM (4-Arm PEG-SCM, 220 mg, 0.02 mmole, 1 eq.,MW: 11,000 Da) was dissolved in 0.55 mL of dichloromethane in a 3 mLvial under Argon. Propargylamine (8.8 mg, 0.16 mmole, 8 eq.) andtriethylamine (16.2 mg, 0.16 mmole, 8 eq.) were then added into thevial. The vial was closed with a rubber septum and the solution wasstirred at room temperature under Argon for 18 h. The DCM solution wasthen precipitated into diethylether (10 mL) in a 20 mL vial. 3 mL vialwas rinsed with 0.25 mL of DCM and this portion was also precipitatedinto the diethylether. The solution was filtered using a 150 mm Whatmanfilter paper. The polymer was dissolved in 2 mL of isopropanol at 50° C.and the solution was cooled down to room temperature. The precipitatewas filtered using a 30 mL glass sintered frit and dried under highvacuum overnight (18 h) to give 203 mg of the final polymer (yield:95%). ¹H NMR of the final polymer shows that 4-Arm PEG-SCM chemicalshifts at 2.82 ppm (s, 4H, NCOCH₂CH₂CO) and 4.48 ppm (s, 2H, OCH₂COO)completely disappeared and new peaks at 2.24, 4.02, and 4.09 ppmappeared for the new polymer. ¹H NMR (CDCl₃, 500 MHz) δ: 2.24 (s, 1H,C≡CH), 3.59 (m, CH₂ (PEG)), 4.02 (s, 2H, OCH₂CONH), 4.09 (dd, 2H,CH2C≡CH).

Example 10—Preparation of 4-Arm PEG-acetyl-Rotigotine

Azidoacetyl rotigotine from example 3 (15.9 mg, 0.016 mmole, 1.6 eq.)was dissolved in 3 mL of THF in a vial. 4-Arm PEG-acetylene (110 mg,0.01 mmole, 1 eq., MW: 11,000 Da) was added and mixture was stirred todissolve the polymer completely. Copper (I) iodide (3.1 mg, 0.016 mmole,1.6 eq.) and triethylamine (1.6 mg, 2.21 μL, 0.016 mmole, 1.6 eq.) wereadded to give a clear green color solution. The resulting solution wasstirred at 45° C. under Argon blanket for 17 h. The cloudy mixture(yellow-brownish) was cooled down to room temperature and filtered usinga 0.2 μm PTFE syringe filter. The filtrate was stirred with 2 mL of 0.1N HCl resulting in a slightly cloudy yellow mixture (pH: 2.5). THF wasremoved using a rotary evaporator at 28° C. The resulting aqueoussolution (cloudy) was passed through a Dowex column (10 g, 15 mL). 60 mLof aqueous solution was collected. The solution was then passed througha column packed with 10 g of Amberlite IR-120H (15 mL) resulting in 150mL of aqueous solution. The solution was saturated with NaCl (15 g) andextracted with DCM three times (3×50 mL). Organic layers were separated,combined, dried over Na₂SO₄ (10 g), filtered and concentrated down to0.5 mL and then precipitated into diethylether (20 mL) in a 50 mLbeaker. The precipitate was filtered on a 15 mL glass frit and driedunder high vacuum overnight to give 95 mg of the final product (yield:78%)

¹H NMR (CDCl₁₃, 500 MHz) δ: 0.97 (3H, —NCH₂CH₂CH₃); 1.86 (total of 3H,—NCH₂CH₂CH₃ and —NCHCH₂CH₂C—); 2.51 (1H, —NCHCH₂CH₂C—); 2.79-3.49 (totalof 11H, rest of the aliphatic CH₂ and CH peaks); 3.58 (m, CH₂ (PEG)),3.97 (s, 2H, OCH₂CONH), 4.56 (t, 2H, triazole-CH₂NHCO), 5.39 (s,triazole-CH₂COO); 6.70-7.03 (3H, CH peaks of1,2,3,4-tetrahydronaphtalene); 6.93-7.42 (3H, CH peaks of 2-thiophene);7.68-7.83 (d, CH peak of triazole).

Example 11—Coupling of 4-Arm PEG-acetylene (10K) to AzidopropylRotigotine

95.0 mg of azidopropyl rotigotine.TFA (0.18 mmole) was dissolved in 20mL of THF in a 50 mL one-neck round-bottom flask and 330 mg of 4-ArmPEG-acetylene (Creative PEGWorks, ZQ9214,) (0.03 mmole, MW: 11,000g/mole) was added into the flask and mixture was stirred to dissolve thepolymer (brown mixture). 9.3 mg of copper (I) iodide (0.048 mmole) and6.63 μL of triethylamine (4.8 mg, 0.048 mmole) were added to give aclear brown color solution. The resulting solution was stirred at 45° C.under Argon blanket for 17 h. The brown mixture was cooled down to roomtemperature and filtered through a 0.2 μM PTFE filter. The filtrate wasstirred with 6 mL of 0.1 N HCl resulting in a brown mixture (pH 2.5 bypH paper). THF was removed using a rotary evaporator at 28 ° C. Theresulting cloudy aqueous solution was passed through a column packedwith Dowex (10 mL, M4195, Supelco, 1844261) at the top and 20 g ofAmberlite IR-120 (30 mL, Fluka, BCBF3074V) at the bottom resulting in200 mL of aqueous solution. The solution was saturated with 20 g of NaCland extracted with 50 mL of DCM three times (3×50 mL). The organiclayers were separated, combined, dried over 20 g of Na₂SO₄, filtered,concentrated down to 2 mL and precipitated into 40 mL of diethylether ina 50 mL beaker. The polymer was filtered and dried under high vacuum togive 310 mg of the final product in 81% yield.

¹H NMR (CDCl₃, δ, ppm, TMS): 1.03 (3H, —NCH₂CH₂CH₃); 1.8-3.6 (total of17H, aliphatic CH and CH₂ peaks of rotigotine; 2.56 (2H,—OCOCH₂CH₂-triazole); 3.41 (—C(CH₂O)₄); 3.64 (1000H, —OCH₂CH₂O—); 4.71(2H, —OCH₂-triazole); 4.76 (2H, —OCOCH₂CH₂-triazole); 6.88-7.21 (6H, —CHpeaks of 1,2,3,4-tetrahydronaphtalene and —CH peaks of 2-thiophene);7.76 (1H, —CH peak of triazole).

Example 12: Coupling of 4-Arm PEG-acetylene (20K) to AzidopropylRotigotine

126.2 mg of azidopropyl rotigotine. TFA (ZH-27-9P) (0.24 mmole) wasdissolved in 40 ml of THF in a 50 one-neck round-bottom flask and 624 mgof 4-Arm PEG-acetylene (Creative PEGWorks, ZQ9216) (0.03 mmole, MW:20,800 g/mole) was added into the flask and mixture was stirred todissolve the polymer completely (yellow solution). 9.63 mg of copper (I)iodide (0.048 mmole) and 6.60 μL of triethylamine (4.8 mg, 0.048 mmole)were added to give a clear yellow color solution. The resulting solutionwas stirred at 45° C. under Argon blanket for 40 h. The reaction wastopped after 40 h of stirring. The solution was filtered through a 045μM PTFE filter. The filtrate was stirred with 12 mL of 0.1 N HClresulting in a brown mixture (pH 2.5 by pH paper). THF was removed usinga rotary evaporator at 28° C. The resulting cloudy aqueous solution waspassed through a column packed with Dowex (20 mL, M4195, Supelco,1844261) at the top and 40 g of Amberlite 1R-120 (60 mL, Fluka,BCSF3074V) at the bottom resulting in 400 mL of aqueous solution. Thesolution was saturated with 40 g of NaCl and extracted with 50 mL of DCMthree times (3×50 mL). The organic layers were separated, combined,dried over 20 g of Na₂SO₄, filtered and concentrated down to 4 mL. TheDCM solution was then precipitated into 80 mL of diethylether in a 100mL beaker. The solvent was decanted and the polymer was dried under highvacuum to give 582 mg of the final product in 86% yield.

¹11 NMR (CDCl₃, 6, ppm, TMS): 1.03 (3H, —NCH₂CH₂CH₃); 1.8-3.6 (total of17H, aliphatic CH and CH₂ peaks of rotigotine; 2.56 (2H,—OCOCH₂CH₂-triazole); 3.41 (2H, —C(CH₂O)₄); 3.64 (1000H, —OCH₂CH₂O—);4.69 (2H, —OCH₂-triazole); 4.74 (2H, —OCOCH₂CH₂-triazole); 6.88-7.21(6H, —CH peaks of 1,2,3,4-tetrahydronaphtalene and —CH peaks of2-thiophene); 7.71 (1H, —CH peak of triazole).

Example 13—Preparation of 2-Arm PEG Acetylene (10K)

1.05 g of SCM-PEG-SCM (0.1 mmole, MW: 10,500 g/mole) was dissolved in2.5 mL of dichloromethane (DCM) in a 10 mL vial under Argon and 25.6 μl,of propargylamine (22 mg, 0.4 mmole) and 56.5 μL of triethylamine (41mg, 0.4 mmole) were then added into the vial. The vial was closed with arubber septum and the solution was stirred at room temperature underArgon for 18 h. The DCM solution was then precipitated into 50 mL ofdiethylether in a 100 mL beaker. 10 mL vial was rinsed with 1 mL of DCMand this portion was also precipitated. The solution was filtered usinga 150 mm Whatman filter paper. The filtered polymer was redissolved in50 mL of isopropanol at 50° C. and cooled down to room temperature. Thepolymer was recrystallized upon cooling. The polymer was filtered using30 mL glass sintered frit and dried under high vacuum overnight to give1.0 g of the final polymer in 96% yield (BD-23-86-1). ¹H NMR (CDCl₃, δ,ppm, TMS): 2.24 (1H, —CONHCH₂C≡CH), 3.64 (920H, —OCH₂CH₂O—), 4.02 (2H,—OCH₂CONHCH₂C≡CH), 4.10-4.15 (2H, —CONHCH₂C—CH).

Example 14: Coupling of 2-Arm PEG-acetylene (10K) to Rotigotine3-azidopropionate

47.3 mg of azidopropyl rotigotine. TFA (0.09 mmole) was dissolved in 20ml of THF in a 50 mL one-neck round-bottom flask and 315 mg ofacetylene-PEG-acetylene (0.03 mmole) was added into the flask andmixture was stirred to dissolve the polymer completely (clear colorlesssolution). 9.3 mg of copper (I) iodide (0.048 mmole) and 6.63 μL oftriethylamine (4.8 mg, 0.048 mmole) were then added into the flask togive a clear green color solution. The resulting solution was stirred at45° C. under Argon blanket for 20 h. The green color mixture was cooleddown to room temperature and filtered using a 0.2 μm PTFE syringefilter. The filtrate was stirred with 6 mL of 0.1 N HCl resulting in ayellow mixture (pH 2.5 by pH paper). THF was removed using a rotaryevaporator at 28° C. The resulting cloudy aqueous solution was passedthrough a column packed with 10 mL of Dowex (M4195, Supelco, 1844261) atthe top and 20 g of Amberlite IR-120 (30 mL, Fluka, BCBF3074V) at thebottom resulting in 200 mL of aqueous solution. The aqueous solution wassaturated with 20 g of NaCl and extracted with 50 mL of DCM three times(3×50 mL). The organic layers were separated, combined, dried over 20 gof Na₂SO₄, filtered, concentrated down to 2 mL and precipitated into 40mL of diethylether in a 50 mL beaker. The precipitated polymer wasfiltered and dried under high vacuum to give 250 mg of the final productin 73% yield.

¹H NMR (CDCl₃, δ, ppm, TMS): 1.03 (3H, —NCH₂CH₂CH₃); 1.8-3.6 (total of17H, aliphatic CH and CH₂ peaks of rotigotine; 2.63 (2H,—OCOCH₂CH₂CH₂-triazole); 3.64 (920H, —OCH₂CH₂O—); 4.02 (2H,—OCH₂CONHCH₂C≡CH); 4.61 (2H, —CONHCH₂-triazole); 4.76 (2H,—OCOCH₂CH₂-triazole); 6.87-7.21 (6H, —CH peaks of1,2,3,4-tetrahydronaphtalene and —CH peaks of 2-thiophene); 7.75 (—CHpeak of triazole); 7.81 (1H, —CONH—).

Example 15—Preparation of 4-Arm PEG Rotigotine Glycine Ester (10K)

Glycine-Rotigotine synthesis: Rotigotine HCl (1.2 g, 3.41 mmol) andBoc-Glycine OH (1.195 g, 6.82 mmol) were dissolved in dichloromethane(150 ml) to give a suspended solution. After the addition of DMAP (0.625g, 5.11 mmol) and DCC (1.407 g, 6.82 mmol), the mixture was stirred for16 hours at room temperature. The mixture was filtered using a filterpaper and the filtrate was quenched with 51 mL of 0.1 N HCl (5.11 mmol).Two layers were separated and the aqueous phase was extracted with 7 mLof dichloromethane. The combined organic phases were washed with waterand then with brine, dried over Na₂SO₄, filtered, concentrated using arotary evaporator, and dried in vacuo to give a crude as pale yellowsolids. The crude material was stirred with diethyl ether (50 mL) for 30minutes, filtered on a glass frit, rinsed with diethyl ether, and driedin vacuo to give a pale yellow powder as a desired productBoc-Gly-Rotigotine. HCl (1.258 g, 75% yield).¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed peaks at 1.04 ppm (t,3H, —CH₂CH₂CH₃), 1.47 ppm (s, 9H, —NHBoc), 1.96 ppm (m, 2H), 2.06 ppm(m, 1H), 2.60 ppm (m, 2H), 2.93 ppm (m, 1H), 3.04 ppm (m, 1H), 3.13 ppm(m, 1H), 3.26 ppm (m, 2H), 3.40 ppm (m, 2H), 3.52 ppm (m, 1H), 3.66 ppm(m, 2H), 4.17 ppm (d, 2H, —NHCH₂C(═O)—), 5.08 ppm (s, 1H, —C(═O)NHCH₂—),6.95 ppm (m, 3H, aromatic), 7.06 ppm (t, 1H, thiophenyl), and 7.20 ppm(m, 2H, thiophenyl).The Boc-Gly-Rotig HCl was deprotected by first dissolving 1.258 g (2.55mmol) in dichloromethane (64 ml). After addition of trifluoroacetic acid(9.83 ml, 128 mmol), the reaction mixture was stirred for 1 hour at roomtemperature and then all the volatiles were removed using a rotaryevaporator. The residue (dark yellow) was redissolved in methanol andprecipitated by adding into diethyl ether (40 mL). The pale yellowprecipitates were filtered using a glass fit and dried to giveGly-Rotigotine. 2TFA (1.140 g, 79% yield).¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed peaks at 0.98 ppm (d,3H, —CH₂CH₂CH₃), 1.72 ppm (m, 1H), 1.83 ppm (m, 2H), 2.33 ppm (m, 1H),2.51 ppm (m, 2H), 2.80 ppm (m, 1H), 3.00 ppm (m, 21-1), 3.12 ppm (m,2H), 130 ppm (m, 3H), 3.73 ppm (m, 1H), 4.03 ppm (q, 2H, NH₂CH₂C(═O)O—),6.80 ppm (d, 1H, aromatic), 6.92 ppm (m, 2H, aromatic), 6.99 ppm (d, 1H,thiophenyl), 7.08 ppm (t, 1H, thiophenyl), and 7.17 ppm (d, 1H,thiophenyl).

4-arm PEG-SCM 10K (2.02 g, 0.165 mmol) and Gly-Rotigotine. 2TFA (0.373g, 0.658 mmol) were dissolved in dichloromethane (16.5 ml). TEA (0.229ml, 1.645 mmol) was added to give a yellow clear solution. Afterstirring for 16 hours at room temperature, the mixture was quenched with16 mL of 0.1N HCl solution and charged with 1.6 g of NaCl (10 w/v % forwater). Two layers were separated and the aqueous phase was extractedwith 16 mL of dichloromethane. The combined organic phases were driedover Na₂SO₄, filtered, and concentrated. The crude extract was dissolvedin 40 mL of water and passed through Amberlite (IR120H) column to removeall the small molecules. The collected aqueous solution was stirred with50 mL of dichloromethane and charged with 10.5 g of NaCl (15 w/v % ofwater). Two layers were separated and the aqueous phase was extractedwith additional 50 mL of dichloromethane. The combined organic phaseswere dried over Na₂SO₄, filtered, concentrated, and dried in vacuo togive the desired product 4-arm PEG-Gly-Rotigotine. HCl 10K (1.89 g, 85%yield).¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed the polymer backbonepeaks at 3.64 ppm (m, 4H, —(OCH₂CH₂)_(n)—) and other major peaks at 1.04ppm (d, 3H, —CH₂CH₂CH₃), 6.96 ppm (m, 3H, aromatic), 7.05 ppm (t, 1H,thiophenyl), 7.20 ppm (m, 2H, thiophenyl), and 7.80 ppm (m, 1H,triazole). The average number of rotigotine molecules on each polymerwas determined as 3.1 by ¹H NMR analysis.

Example 16—Preparation of 4-Arm PEG Rotigotine Glycine Ester (20K)

The glycine-rotigotine. 2TFA salt was prepared as described in example16. The 4-arm PEG-SCM 20K (2.007 g, 0.098 mmol) and Gly-Rotigotine. 2TFA(0.222 g, 0.393 mmol) were dissolved in dichloromethane (9.8 ml). TEA(0.137 ml, 0.981 mmol) was added to give a yellow clear solution. Afterstirring for 16 hours at room temperature, the mixture was quenched with9.8 mL of 0.1N HCl solution and charged with 1.0 g of NaCl (10 w/v % forwater). Two layers were separated and the aqueous phase was extractedwith 10 mL of dichloromethane. The combined organic phases were driedover Na₂SO₄, filtered, and concentrated. The crude extract was dissolvedin 40 mL of water and passed through Amberlite (IR120H) column to removeall the small molecules. The collected aqueous solution was stirred with50 mL of dichloromethane and charged with 10.5 g of NaCl (15 w/v % ofwater). Two layers were separated and the aqueous phase was extractedwith 40 mL of dichloromethane. The combined organic phases were driedover Na₂SO₄, filtered, concentrated, and dried in vacuo to give thedesired product 4-arm PEG-Gly-Rotigotine. HCl K (1.58 g, 74% yield).¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed the polymer backbonepeaks at 3.64 ppm (m, 4H, —(OCH₂CH₂)_(n)—) and other major peaks at 1.03ppm (d, 3H, —CH₂CH₂CH₃), 6.95 ppm (m, 3H, aromatic), 7.06 ppm (t, 1H,thiophenyl), 7.20 ppm (m, 2H, thiophenyl), and 7.81 ppm (m, 1H,triazole). The average number of rotigotine molecules on each polymerwas determined as 2.53 by ¹H NMR analysis.

Example 17—Preparation of H-[(Ethyl-Tiagabine)₁₀(EOZ)₁₉₀ ]-COOH 20K byAttachment of Tiagabine 3-azidoacetate to Polyoxazoline 10 Pendent Acid20K

In a 250 mL round bottom flask, tiagabine (2.00 gm, 5.33 mmol), 4-DMAP(658 mg, 5.33 mmol) were dissolved in anhydrous ACN (100 mL). ACN wascompletely evaporated by rotary-evaporation. DCM (100 mL) was added todissolve the residual, which was allowed to stir under argon. To thesolution 2-bromoethanol (1.17 mL, 15.98 mmol) and DCC were added (1.16gm, 5.59 mmol). The solution was allowed to stir at room temperature forovernight. The pink solution turned cloudy. Following overnight ofreaction, the reaction mixture was analyzed by reversed phase HPLC,which indicated 98% of conversion to 2-bromoethyl tiagabine. Thereaction mixture was filtered; the pink filtrate was washed twice with0.1 N HCl using 100 mL each time in a separatory funnel. Following phaseseparation, DCM phase was dried over anhydrous sodium sulfate (100 gm).The mixture was filtered through glass frit. The filtrate wasconcentrated to dryness by rotary-evaporation. The residual wasdissolved in DCM (30 mL). White precipitate was filtered off. Thefiltrate was concentrated to 10 mL, which was then added into hexanes(300 mL) to precipitate. The solid was collected in a glass fritfollowing filtration, and dried in vacuum to provide compound 2a assolid powder (2.1 gm, Yield: 72%). NMR analysis of 2a in deuteratedchloroform showed the relevant peaks at 4.405 ppm (t, 2H, BrCH₂CH₂O—);3.496 ppm (t, 2H, BrCH₂CH₂O—); 7.251 ppm (d, 1H, —S—CH═CH—); 7.095 ppm(d, 1H, —S—CH═CH—); 6.889 ppm (d, 1H, —S—CH═CH—); 6.774 ppm (d, 1H,—S—CH═CH—); 5.965 ppm (t, 1H, ═CHCH₂—); 2.030 ppm (s, 3H, CH₃—); 1.983ppm (s, 3H, CH₃—). 1.455 ppm-3.653 ppm (m, 16H, CH₃— and BrCH₂— notincluded).

To 2-Bromoethyl Tiagabine·HCl salt (2a) (2.00 gm, 3.70 mmol) in a 100 mLround bottom flask with 20 mL of anhydrous DMF, TEA (1.11 mL, 7.98 mmol)and NaN₃ (262 mg, 3.99 mmol) were added into the solution. The solutionwas allowed to stir at 40° C. with an oil bath under argon atmosphere.Following overnight of stirring, DMF was evaporated at 40° C. undervacumm by rotary-evaporation. Ethyl acetate (100 mL) and 0.1 N HCl (60mL) was added to the mixture, stirred, and then transferred into aseparatory funnel. Following phase separation, the aqueous phase wasextracted by ethyl acetate again (100 mL). The ethyl acetate layer wascombined, washed with 0.1 N HCl (20 mL). The ethyl acetate layer wasthen dried over sodium sulfate (100 gm). Following filtration, the clearfiltrate was concentrated to 20 mL in a 250 mL round bottom flask byrotary evaporation. To the mixture hexanes (200 mL) was added toprecipitate the product. The solid was collected into a glass fritfollowing filtration, and dried overnight in vacuum, which provide 1.38gm of crude product in solid form. The crude product (1.0 gm) wasre-dissolved in a solution of 0.1% TFA in ACN (24 mL), and then 0.1% TFAin water (96 mL). White precipitate in the mixture was filtered off. Thefiltrate was then purified by reversed phase chromatography with aSunFire Prep C8 OBD 30/250 Column from Waters using a UV detector atwavelength 214 nm at a flow rate of 20 mL/min. 0.1% TFA in water (Mobilephase A) and 0.1% TFA in ACN (Mobile phase B) were used as mobile phasesfor the purification. The column was equilibrated with 20% B. Followingloading of the crude product, the column was initially elutedisocratically with 20% of mobile phase B. The gradient was ramped to 35%mobile phase B in 15 minutes, and then eluted isocratically with 35% ofmobile phase B. The product fraction was collected when the column waseluted with 35% mobile phase B. The solution was evaporated by rotaryevaporation to remove ACN. The remaining aqueous solution was extractedby DCM (3×120 mL). Following phase separation, DCM phase was dried overanhydrous sodium sulfate (100 gm). The solid was filtered off, and thefiltrate was concentrated by rotary-evaporator to near dryness, and thendried in vacuum to provide compound 2b as viscous oil (0.89 gm). NMRanalysis in deuterated chloroform showed the relevant peaks at 4.272 ppm(m, 2H, N₃CH₂CH₂O—); 3.483 ppm (t, 2H, N₃CH₂CH₂O—); 7.251 ppm (d, 1H,—S—CH═CH—); 7.091 ppm (d, 1H, —S—CH═CH—); 6.883 ppm (d, 1H, —S—CH═CH—);6.769 ppm (d, 1H, —S—CH═CH—); 5.934 ppm (t, 1H, ═CHCH₂—); 2.021 ppm (s,3H, CH₃—); 1.972 ppm (s, 3H, CH₃—). 1.455 ppm-3.733 ppm (m, 16H, CH₃—and BrCH₂— not included). HPLC purity 99%.

H-[(PtynOZ)₁₀(EOZ)₁₉₀]-T-PA (1.13 gm, 0.0577 mmol) was dissolved in THF(25 mL) in a 100 mL RB flask with 2-azidoethyl tiagabine·HCl salt (2b)(344.5 mg, 0.635 mmol). The solution was protected under argon. CuI(44.2 mg, 0.231 mmol) was then added to the flask, followed by immediateaddition of TEA (0.12 mL, 0.866 mmol). The solution, which turnedgreenish, was stirred at 45° C. for overnight under argon atmosphere.The solution was filtered to remove solid. 0.1 N HCl (20 mL) was addedinto the filtrate. THF in the mixture was then evaporated byrotary-evaporator. The remaining aqueous solution (20 mL) was thenloaded to a column (2 cm i.d.) packed with Dowex® M4195 media (20 gm)over silica gel 60 (14 gm), which was equilibrated in 2 mM HCl, toremove copper ion. The column was eluted with 2 mM HCl until noPOZ-Tiagabine conjugate was retained on the column. To remove lowmolecular weight tiagabine related species (tiagabine and 2-azidoethyltiagabine), the collected eluate (175 mL) was then applied to a columnpacked with Amberlite TR-120 (41 gm) resin, followed by elution with 2mM HCl until POZ-Tiagabine conjugate completely eluted. NaCl (15 gm) wasadded to the collected eluate (300 mL) to make 5% brine. The solutionwas extracted with DCM (3×100 mL). Following phase separation, the DCMphases were pooled, and dried over anhydrous sodium sulfate (100 gm) forone hour. The mixture was filtered through a glass fit to remove sodiumsulfate. The filtrate was concentrated to 25 mL by rotary evaporation,and then precipitated in 500 mL of diethyl ether. The precipitate wascollected when the mixture was filtered through a glass frit, and thendried in vacuum, which yield 1.1 gm of polyoxazoline pendent 2-ethyltiagabine (4) as white powder. HPLC analysis indicated thatPOZ-Tiagabine conjugate did not contain free tiagabine, or 2-azidoethyltiagabine. NMR analysis of polyoxazoline pendent 2-ethyl tiagabine indeuterated chloroform showed the relevant peaks at 7.548 ppm (m, illresolved, nH, ═CH—N); 7.256 ppm (d, nH, —S—CH═CH—); 7.087 ppm (d, nH,—S—CH═CH—); 6.888 ppm (d, nH, —S—CH═CH—); 6.772 ppm (d, nH, —S—CH═CH—);5.958 ppm (t, nH, ═CHCH₂—); 4.761 ppm (t, ill resolved, 2nH,—C(═O)CH₂CH₂CH₂—); 4.494 ppm (m, 2nH, N₃CH₂CH₂O—); 3.449 ppm, 2.406 ppmand 1.120 ppm (polymer backbone). Average number (n) of pendenttiagabine molecule on each POZ was 9.4.

Example 18—Preparation of H-[(Propyl-Tiagabine)₁₀(EOZ)₁₉₀]-COOH 20K byAttachment of Tiagabine 3-azidopropionate to Polyoxazoline 10 PendentAcid 20K

In a 250 mL round bottom flask, tiagabine (2.00 gm, 5.33 mmol), 4-DMAP(658 mg, 5.33 mmol) were dissolved in anhydrous ACN (100 mL). ACN wascompletely evaporated by rotary-evaporation. DCM (100 mL) was added todissolve the residual, which was allowed to stir under argon. To thesolution 3-bromo-1-propanol (1.49 mL, 15.98 mmol) and DCC were added(1.16 gm, 5.59 mmol). The solution was allowed to stir at roomtemperature for overnight. The pink solution turned cloudy. Followingovernight of reaction, the reaction mixture was analyzed by reversedphase HPLC, which indicated 96% of conversion to 3-bromopropyl tiagabineester. The reaction mixture was filtered; the pink filtrate was washedtwice with 0.1 N HCl using 100 mL each time in a separatory funnel.Following phase separation, DCM phase was dried over anhydrous sodiumsulfate. The mixture was filtered through glass frit. The filtrate wasconcentrated to dryness by rotary-evaporation. The residual was furtherdried in vacuum. The residual crude product was re-dissolved in asolution of 0.1% TFA in ACN (42 mL), and then 0.1% TFA in water (78 mL).White precipitate in the mixture was filtered off. The filtrate was thenpurified by reversed phase chromatography with a SunFire Prep C8 OBD30/250 Column from Waters using a UV detector at wavelength 214 nm. 0.1%TFA in water (Mobile phase A) and 0.1% TFA in ACN (Mobile phase B) wereused as mobile phases for the purification. The column was equilibratedwith 35% B. Following loading of the crude product, the column waseluted isocratically with 35% of mobile phase B. The product fractionwas collected and analyzed by reversed phase HPLC. The solution wasevaporated by rotary evaporation to remove ACN. The remaining aqueoussolution was extracted by DCM (3×250 mL). Following phase separation,DCM phase was dried over anhydrous sodium sulfate (100 gm). The solidwas filtered off, and the filtrate was concentrated by rotary-evaporatorto near dryness, and then dried in vacuum to provide compound la asviscous oil (1.83 gm, yield: 56%). NMR analysis in deuterated chloroformshowed the relevant peaks at 4.247 ppm (t, 2H, BrCH₂CH₂CH₂O—); 3.441 ppm(t, 2H, BrCH₂CH₂CH₂O—); 7.253 ppm (d, 1H, —S—CH═CH—); 7.094 ppm (d, 1H,—S—CH═CH—); 6.884 ppm (d, 1H, —S—CH═CH—); 6.771 ppm (d, 1H, —S—CH═CH—);5.932 ppm (t, 1H, ═CHCH₂—); 2.029 ppm (s, 3H, CH₃—); 1.973 ppm (s, 3H,CH₃—). 1.455 ppm-3.668 ppm (m, 16H, CH₃— and BrCH₂— not included). HPLCpurity 98%.To the 3-Bromopropyl Tiagabine Ester·TFA salt (1.80 gm, 2.89 mmol) in a100 mL round bottom flask with 20 mL of anhydrous DMF, TEA (806 μL, 5.78mmol) and NaN₃ (188 mg, 2.89 mmol) were added into the solution. Thesolution was allowed to stir at 40° C. with an oil bath under argonatmosphere. Following overnight of stirring, DMF was evaporated at 40°C. under vacuum by rotary-evaporation. Ethyl acetate (100 mL) and 0.1 NHCl (60 mL) was added to the mixture, stirred, and then transferred intoa separatory funnel. Following phase separation, the aqueous phase wasextracted by ethyl acetate again (100 mL). The ethyl acetate layer wascombined, washed with deionized water (50 mL). The ethyl acetate layerwas then dried over sodium sulfate. Following filtration, the clearfiltrate was concentrated to dryness by rotary evaporation. The residualwas further dried in vacuum to provide compound 1b as viscous oil (1.53gm, Yield: 97%). NMR analysis in deuterated chloroform showed therelevant peaks at 4.190 ppm (t, 2H, N₃CH₂CH₂CH₂O—); 3.388 ppm (t, 2H,N₃CH₂CH₂CH₂O—); 7.253 ppm (d, 1H, —S—CH═CH—); 7.093 ppm (d, 1H,—S—CH═CH—); 6.884 ppm (d, 1H, —S—CH═CH—); 6.771 ppm (d, 1H, —S—CH═CH—);5.937 ppm (t, 1H, ═CHCH₂—); 2.021 ppm (s, 3-H, CH₃—); 1.973 ppm (s, 3H,CH₃—); 1.457 ppm-3.683 ppm (m, 16H, CH₃— and N₃CH₂— not included). HPLCpurity 92%.

H-[(PtynOZ)₁₀(EOZ)₁₉₀]-T-PA (1.13 gm, 0.0577 mmol) was dissolved in THF(25 mL) in a 100 mL RB flask with 3-Azidopropyl Tiagabine Ester·HCl salt(338.7 mg, 0.635 mmol). The solution was protected under argon. CuI(44.2 mg, 0.231 mmol) was then added to the flask, followed by immediateaddition of TEA (0.12 mL, 0.866 mmol). The solution, which turnedgreenish, was stirred at 45° C. for overnight under argon atmosphere.The greenish solution was filtered to remove solid. 0.1 N HCl (20 mL)was added into the filtrate. THF in the mixture was then evaporated byrotary-evaporator. The remaining aqueous solution (20 mL) was thenloaded to a column (2 cm i.d.) packed with Dowex® M4195 media (20 gm)over silica gel 60 (14 gm), which was equilibrated in 2 mM HCl, toremove copper ion. The column was eluted with 2 mM HCl until noPOZ-Tiagabine conjugate was retained on the column To remove lowmolecular weight tiagabine related species (tiagabine and 3-azidopropyltiagabine ester), the collected eluate (175 mL) was then applied to acolumn packed with Amberlite IR-120 (41 gm) resin, followed by elutionwith 2 mM HCl until POZ-Tiagabine conjugate completely eluted. NaCl (15gm) was added to the collected eluate (300 mL) to make 5% brine. Thesolution was extracted with DCM (3×100 mL). Following phase separation,the DCM phases were pooled, and dried over anhydrous sodium sulfate (100gm) for one hour. The mixture was filtered through a glass frit toremove sodium sulfate. The filtrate was concentrated to 25 mL by rotaryevaporation, and then precipitated in 500 mL of diethyl ether. Theprecipitate was collected when the mixture was filtered through a glassfrit, and then dried in vacuum, which yield 1.1 gm of white powder. HPLCanalysis indicated that POZ-Tiagabine conjugate did not contain freeTiagabine, or 3-Azidopropyl Tiagabine Ester. NMR analysis in deuteratedchloroform showed the relevant peaks at 7.55 ppm (m, ill resolved, nH,═CH—N), -); 7.258 ppm (d, nH, —S—CH═CH—) ); 7.093 ppm (d, nH,—S—CH═CH—); 6.881 ppm (d, nH, —S—CH═CH—); 6.769 ppm (d, nH, —S—CH═CH—);5.964 ppm (t, nH, ═CHCH₂—); 4.425 ppm (t, ill resolved, 2nH,—C(═O)CH₂CH₂CH₂—) ); 3.463 ppm, 2.406 ppm and 1.120 ppm (polymerbackbone).

Example 19—Preparation of H-[(PEG3-Tiagabine)₁₀(EOZ)190]COOH 20K byAttachment of 2-[2-(2-Azidoethoxy)ethoxy]ethyl Tiagabine Ester toPolyoxazoline 10 Pendent Acid 20K

In a 100 mL round bottom flask, tiagabine (786 mg, 2.092 mmol, 1.0equiv.), 4-DMAP (258 mg, 2.092 mmol, 1.0 equiv.), and-[2-(2-Azidoethoxy)ethoxy]ethanol (733 mg, 4.185 mmol, 2.0 equiv.) weredissolved in anhydrous acetonitrile (CAN, 40 mL). ACN was completelyevaporated by rotary-evaporation at 25° C. Anhydrous dichloromethane(DCM, 35 mL) was added to dissolve the residue and stirred in an argonatmosphere. To this solution DCC (458 mg, 2.197 mmol, 1.05 equiv.) wasadded. The solution was allowed to stir at room temperature overnight.The reaction mixture was next filtered to remove solid precipitate andthe pink colored DCM filtrate was washed with 0.1 N HCl (2×50 mL) in aseparatory funnel. Following phase separation, the DCM phase was driedover anhydrous sodium sulfate, filtered and then concentrated to drynessby rotary evaporation. The residue was further dried under vacuum andthe resultant product was 1.35 gm of crude2-[2-(2-Azidoethoxy)ethoxy]ethyl Tiagabine Ester. This crude powder wasnext dissolved in 0.1% TFA in ACN (35 mL), followed by addition of 0.1%TFA in water (65 mL). The mixture was filtered through a glass frit toremove white precipitate. The filtrate was further filtered through a0.45 um membrane and then purified by preparative reverse phasechromatography using a SunFire Prep C8 OBD 30/250 Column (Waters Corp)and a UV detector set at a wavelength of 214 nm. The elution media usedin the purification was 0.1% TFA in water (Mobile phase A) and 0.1% TFAin ACN (Mobile phase B). The column was equilibrated with 35% B.Following loading of the crude product, the column was elutedisocratically with 35% of mobile phase B. The eluted product fractionwas evaporated by rotary evaporation to remove ACN. The remainingaqueous solution was then extracted with DCM (3 times×250 mL). Followingphase separation each time, DCM phase was collected and dried overanhydrous sodium sulfate (100 gm). The solid was filtered off, and thefiltrate was concentrated by rotary-evaporation to near dryness, andthen dried under vacuum to yield 2-[2-(2-Azidoethoxy)ethoxy]ethylTiagabine Ester as a viscous oil (567 mg, yield: 42%).The product was analyzed by reverse phase HPLC to confirm purity of 98%.NMR analysis in deuterated chloroform showed the relevant peaks at 4.255ppm (t, 2H, —C(═O)OCH₂CH₂O—); 3.652-3.703 ppm (m, 4×2H, —OCH₂CH₂O—,—C(═O)OCH₂CH₂O—, —OCH₂CH₂N₃); 3.387 ppm (t, 2H, —CH₂N₃); 7.249 ppm (d,1H, —S—CH═CH—); 7.089 ppm (d, 1H, —S—CH═CH—); 6.879 ppm (d, 1H,—S—CH═CH—); 6.767 ppm (d, 1H, —S—CH═CH—); 5.932 ppm (t, 1H, ═CHCH₂—);2.029 ppm (s, 3H, CH₃—); 1.973 ppm (s, 3H, CH₃—). 1.455 ppm-3.550 ppm(m, 13H).

H-[(PtynOZ)₁₀(EOZ)₁₉₀]-T-PA (1.65 gm, 0.0847 mmol) was dissolved intetrahydrofuran (THF, 35 mL) in a 100 mL RB flask with2-[2-(2-Azidoethoxy)ethoxy]ethyl Tiagabine Ester (561 mg, 0.847 mmol).The solution was mixed in an argon atmosphere. Copper Iodide (CuI, 65mg, 0.339 mmol) was then added to the flask, followed by immediateaddition of triethylamine (TEA, 0.18 mL, 1.270 mmol). The solution,which turned greenish, was stirred at 45° C. for overnight under argonatmosphere. The greenish solution was then filtered to remove any solidresidue, and 0.1 N HCl acid (30 mL) was then added to the filtrate. TheTHF in the mixture was then evaporated by rotary-evaporator. Theremaining aqueous solution (30 mL) was then loaded to a column (2 cmi.d.) packed with Dowex® M4195 media (30 gm) over silica gel 60 (20 gm),which was equilibrated in 2 mM HCl, to remove any soluble copper ionspecies. The column was eluted with 2 mM HCl until no POZ-Tiagabineconjugate was retained on the column. To remove low molecular weightfree tiagabine and unreacted 2-[2-(2-Azidoethoxy)ethoxy]ethyl TiagabineEster species, the collected eluent (256 mL) was loaded onto a columnpacked with Amberlite IR-120 (60 gm) resin, and then eluted with 2 mMHCl acid. To the aqueous solution (400 mL) containing the desiredPOZ-Tiagabine conjugate, was added NaCl (20 gm) to make a brine solutionwith approximately 5% salt. This solution was extracted with DCM (3times×145 mL). Following phase separation, the DCM phases were pooled,and dried over anhydrous sodium sulfate (145 gm) for one hour. Themixture was filtered through a glass frit to remove sodium sulfate. Thefiltrate was concentrated to 30 mL by rotary evaporation, and thenprecipitated in 650 mL of diethyl ether. The precipitate was collectedwhen the mixture was filtered through a glass frit, and then dried invacuum, which yield 1.5 gm of white powder. HPLC analysis showed thatthe desired POZ-Tiagabine conjugate did not contain free Tiagabine, orunreacted 2-[2-(2-Azidoethoxy)ethoxy]ethyl Tiagabine ester. NMR analysisin deuterated chloroform showed the relevant peaks at 7.72 ppm (m, illresolved, nH, ═CH—N); 7.258 ppm (d, nH, —S—CH═CH—); 7.093 ppm (d, nH,—S—CH═CH—); 6.884 ppm (d, nH, —S—CH═CH—); 6.769 ppm (d, nH, —S—CH═CH—);5.962 ppm (t, nH, ═CHCH₂—); 4.575 ppm (t, ill resolved, 2nH,—C(═O)CH₂CH₂CH₂—); 3.472 ppm, 2.406 ppm and 1.120 ppm (polymerbackbone).

Example 20—Preparation of H-[(Phenyl-Tiagabine)₁₀(EOZ)₁₉₀]-COOH 20K byAttachment of Tiagabine 3-azido-N-(4-hydroxyphenyl)propanamide Ester toPolyoxazoline 10 Pendent Acid 20K

Succinimidyl azidopropionate: 3-Azidopropionic acid (5.00 gm, purity95.4%, 41.446 mmol, 1.0 eq.) and N-hydroxysuccinimide (NHS, 4.87 gm,41.446 mmol, 1.0 eq.) were dissolved in DCM (150 mL), followed byaddition of DCC (8.64 gm, 41.446 mmol, 1.0 eq). The solution was allowedto stir under argon at room temperature. Following overnight ofreaction, the cloudy mixture was filtered to remove white precipitate.The filtrate was evaporated by rotary evaporation to dryness. Theresidual was dissolved in ACN (100 mL) and any white precipitate presentin ACN was filtered off The filtrate was evaporated to dryness, byrotary evaporation, followed by further drying under vacuum. Theresultant product of succinimidyl azidopropionate was 9.7 gm. NMRanalysis in DMSO-d6 showed the relevant peaks at 3.659 ppm (t, 2H,N₃CH₂—); 3.012 ppm (t, 2H, —N₃CH₂CH₂—); 2.822 ppm (s, 4H, —OSu). Reversephase HPLC purity was 95%.

3-Azido-N-(4-hydroxyphenyl)propanamide: In the next step, 4-aminophenol(1.47 gm, 13.433 mmol, 0.75 eq.) was dissolved in an ACN-water mixedsolvent (1:1 v/v, 60 mL) at 60° C. The solution was transferred into theround bottom flask which contained the succinimidyl azidopropionate(4.00 gm, 17.911 mmol, 1.0 eq.). The solution was allowed to stir at 60°C. under Argon atmosphere. Following overnight of reaction, the mixturewas filtered through a 0.45 μm membrane. The filtrate was evaporated toremove ACN completely and during the process a precipitate was formed inthe remaining aqueous solution. The supernatant was decanted and theresidual precipitate was next washed with DI water (30 mL), decanted,and then redissolved in ACN (30 mL). The solution was placed on a rotaryevaporator and the solvent was evaporated to leave behind a residue thatrequired additional drying under vacuum. The dried product was 0.79 gmof 3-Azido-N-(4-hydroxyphenyl)propanamide. NMR analysis in DMSO-d6showed the relevant peaks at 7.352 ppm (d, 2×1H, phenyl); 6.684 ppm (d,2×1H, phenyl); 3.590 ppm (t, 2H, N₃CH₂CH₂—); 2.552 ppm (t, 2H,N₃CH₂CH₂—). 4-(3-Azidopropanamido)phenyl Tiagabine Ester: In a 250 mLround bottom flask, 3-Azido-N-(4-hydroxyphenyl)propanamide (787 mg,3.641 mmol, 2.0 eq.), tiagabine (684 mg, 1.821 mmol, 1.0 equiv.), 4-DMAP(225 mg, 1.821 mmol, 1.0 equiv.) were dissolved in 19 mL of anhydrousACN (10 mL). ACN was completely evaporated by rotary-evaporation at 28°C. DMF (15 mL) was added to dissolve the residual, which was allowed tostir under argon. To the solution DCC were added (398 mg, 1.912 mmol,1.05 equiv.). The solution was allowed to stir at room temperatureovernight. The reaction mixture was evaporated at 35° C. under vacuum toremove DMF. The residual was dissolved in 0.1% TFA in ACN (35 mL),followed by addition of 0.1% TFA in water (65 mL). The mixture wasfiltered through a glass frit to remove white precipitate. The filtratewas further filtered through a 0.45 um membrane. The filtrate was thenpurified by reversed phase chromatography with a SunFire Prep C8 OBD30/250 Column from Waters using a UV detector at wavelength 214 nm. 0.1%TFA in water (Mobile phase A) and 0.1% TFA in ACN (Mobile phase B) wereused as mobile phases for the purification. The product fraction wascollected and analyzed by reversed phase HPLC. The solution wasevaporated by rotary evaporation to remove ACN. The remaining aqueoussolution was extracted by DCM (3×250 mL). Following phase separation,DCM phase was dried over anhydrous sodium sulfate (100 gm). The solidwas filtered off, and the filtrate was concentrated by rotary-evaporatorto near dryness, and then dried in vacuum to provide4-(3-Azidopropanamido)phenyl Tiagabine Ester as viscous oil (484 mg).NMR analysis in deuterated chloroform showed the relevant peaks at 7.563ppm (d, 2H, phenyl); 6.996 ppm (d, 2H, phenyl); 7.235 ppm (d, 1H,—S—CH═CH—); 7.089 ppm (d, 1H, —S—CH═CH—); 6.874 ppm (d, 1H, —S—CH═CH—);6.768 ppm (d, 1H, —S—CH═CH—); 5.946 ppm (t, 1H, ═CHCH₂—); 3.725 ppm (t,2H, N₃CH₂—); 2.620 ppm (t, 2H, N₃CH₂CH₂—); 2.029 ppm (s, 3H, CH₃—);1.973 ppm (s, 3H, CH₃—). HPLC purity 92%.

H-[(PtynOZ)10(EOZ)₁₉₀]-T-PA (1.29 gm, 0.0659 mmol) was dissolved in THFmL) in a 100 mL RB flask with 4-(3-Azidopropanamido)phenyl TiagabineEster (484 mg, 0.659 mmol) in an argon atmosphere. Copper iodide (CuI,50 mg, 0.264 mmol) was then added to the flask, followed by immediateaddition of triethylamine (TEA, 0.14 mL, 0.989 mmol). The solution,which turned greenish, was stirred at 45° C. for overnight under argonatmosphere. The greenish solution was filtered to remove solid and 0.1 NHCl acid (24 mL) was added to the filtrate. THF in the mixture was thenevaporated by rotary-evaporation and the remaining aqueous solution (24mL) became cloudy. 2 mM HCl acid (26 mL) was added into the aqueousmixture to dissolve the insoluble material and clarify the solution. Thesolution was then loaded onto a column (2 cm i.d.) packed with Dowex®M4195 media (24 gm) over silica gel 60 (16 gm), which was equilibratedin 2 mM HCl, to remove copper ion. The column was eluted with 2 mM HCluntil no POZ-Tiagabine conjugate was retained on the column. To removethe low molecular weight free tiagabine and unreacted4-(3-Azidopropanamido)phenyl Tiagabine Ester, the collected eluent (205mL) was loaded onto a column packed with Amberlite IR-120 (48 gm) resin,and then eluted with 2 mM HCl acid. The eluent (320 mL) was collectedand NaCl (16 gm) was added to it to make a brine solution with 5% salt.The solution was extracted with DCM (3 times×100 mL). Following phaseseparation each time, the DCM phases were collected, pooled, and driedover anhydrous sodium sulfate (100 gm) for one hour. The mixture wasfiltered through a glass frit to remove sodium sulfate. The filtrate wasconcentrated to 30 mL by rotary evaporation, and then precipitated in to400 mL of diethyl ether. The precipitate was collected when the mixturewas filtered through a glass frit, and then dried in vacuum, to yield1.2 gm of white powder.HPLC analysis indicated that POZ-Tiagabine conjugate did not containfree Tiagabine, or 4-(3-Azidopropanamido)phenyl Tiagabine Ester. NMRanalysis in deuterated chloroform showed the relevant peaks at 7.607 ppm(d, ill resolved, 2nH, phenyl); 7.548 ppm (m, ill resolved, nH, ═CH—N);7.245 ppm (d, nH, —S—CH═CH—); 7.091 ppm (d, nH, —S—CH═CH—); 6.942 ppm(d, ill resolved, 2H, phenyl); 6.874 ppm (d, nH, —S—CH═CH—); 6.767 ppm(d, nH, —S—CH═CH—); 5.970 ppm (t, nH, ═CHCH₂—); 4.705 ppm (t, illresolved, 2nH, —C(═O)CH₂CH₂CH₂—); 3.457 ppm, 2.401 ppm and 1.118 ppm(polymer backbone).

Example 21—Coupling of 4-Arm PEG-acetylene (10K) to AzidopropylTiagabine

4arm PEG-Alkyne 10K (1.59 gm, 0.144 mmol from Creative PEGWorks) wasdissolved in 25 mL of THF in a 100 mL RB flask with3-Azidopropyl-Tiagabine Ester·HCl salt (338.7 mg, 0.635 mmol). Thesolution was protected under Ar, and heated to 45° C. to dissolve. CuI(44.2 mg, 0.231 mmol) was then added to the flask, followed by immediateaddition of TEA (120.6 μL, 0.866 mmol). The solution was stirred at 45°C. for overnight under argon atmosphere. The solution was filtered toremove, solid. 0.1 N HCl (20 mL) was added into the filtrate. THF in themixture was then evaporated by rotary-evaporator. The remaining aqueoussolution (20 mL) was then loaded to a column (2 cm i.d.) packed withDowex® M4195 media (20 gm), which was equilibrated in 2 mM HCl, toremove copper ion. The column was eluted with 2 mM HCl until noPEG-Tiagabine conjugate was retained on the column. To remove lowmolecular weight tiagabine related species (tiagabine and 3-azidopropyltiagabine ester), the collected eluate was then applied to a columnpacked with Amberlite IR-120 (41 gm) resin, followed by elution with 2mM HCl until PEG-Tiagabine conjugate completely eluted. NaCl (11 gm) wasadded to the collected eluate (220 mL) to make 5% brine. The solutionwas extracted with DCM (3×100 mL). Following phase separation, the DCMphases were pooled, and dried over anhydrous sodium, sulfate (100 gm)for one hour. The mixture was filtered through a glass frit to removesodium sulfate. The filtrate was concentrated to 3 mL by rotaryevaporation, and then precipitated in diethyl ether (200 mL). Theprecipitate was collected when the mixture was filtered through a glassfit, and then dried in vacuum, which yield 1.4 gm of white powder. NMRanalysis in deuterated chloroform showed the relevant peaks at 7.62 ppm(s, 4H, ═CH—N), -); 7.259 ppm (d, 4H, —S—CH═CH—); 7.096 ppm (d, 4H,—S—CH═CH—); 6.883 ppm (d, 4H, —S—CH═CH—); 6.772 ppm (d, 4H, —S—CH═CH—);5.967 ppm (t, 4H, ═CHCH₂—); 4.440 ppm (t, ill resolved, 8H,—C(═O)CH₂CH₂CH₂—); 3.64 ppm (PEG backbone). Average number of Tiagabinemolecule on each 4arm-PEG was 3.2.

Example 22—Preparation of H-[(Carbamate-Ropinirole)₁₀(EOZ)₁₉₀]-COOH 20Kby Attachment of Ropinirole 3-azidocarbamate to Polyoxazoline 10 PendentAcid 20K

Bromoethyl-N-ropinirolylcarbamate: To a solution of ropinirolehydrochloride (0.558 g, 1.88 mmol) in Dioxane (38 ml) was addedtriethylamine (2.10 ml, 15.1 mmol). After stirring for 5 minutes,2-bromoethyl chloroformate (1.61 ml, 15.1 mmol) was added slowly and themixture was allowed to stir overnight at room temperature. Water (40 mL)was added to give a mixture with pH of 9.5. After stirring overnight,the mixture was stirred with dichloromethane (40 mL) and brine solution(10 mL) for 10 minutes. Two layers were separated and the top layer wasextracted with dichloromethane (40 mL). The combined organic phases weredried over Na₂SO₄, filtered, and concentrated to give dark red coloredthick oil. Further purification was performed by silica gel columnchromatography eluting with dichloromethane/EtOAc (starting from 9:1,4:1, and then 100% EtOAc) to give the desired N-acylated product,bromoethyl-N-ropinirolylcarbamate, as dark red colored oil (0.170 g,22.01% yield).). ¹H NMR (Varian, 500 MHz, 10 mg/mL DMSO-d6, δ): 0.83 (t,J=7.5 Hz, 6H, —CH₂CH₂CH₃), 1.39 (m, 4H, —CH₂CH₂CH₃), 2.39 (t, J=7.5 Hz,4H, —CH₂CH₂CH₃), 2.62 (m, 4H, Pr₂NCH₂CH₂—Ar), 3.80 (s, 2H, —CH₂C(═O)—),3.80 (t, J=5.5 Hz, 2H, —OCH₂CH₂Br), 4.65 (t, 2H, —OCH₂CH₂Br), 7.04 (d, J=8.0 Hz, 1H, Ar H), 7.25 (t, J=8.0 Hz, 1H, Ar H), 7.63 (d, J=8.0 Hz, 1H,Ar H). Azidoethyl-N-ropinirolylcarbamate: To a solution ofbromoethyl-N-ropinirolylcarbamate (0.170 g, 0.414 mmol) in DMF (2 ml)was added sodium azide (0.027 g, 0.414 mmol) to give a clear yellowsolution. After stirring overnight at room temperature, the mixture wasquenched with 1 mL of 0.1N HCl and then diluted with 2 mL of water. Allthe volatiles were removed using a rotary evaporator and the aqueoussolution was extracted twice with dichloromethane (3 mL each). Thecombined organic phases were dried over Na₂SO₄, filtered, andconcentrated to give azidoethyl-N-ropinirolylcarbamate (0.12 g, 78%yield) as thick yellow oil. ¹H NMR (Varian, 500 MHz, 10 mg/mL DMSO-d₆,6): 0.93 (t, J=Hz, 6H, —CH₂CH₂CH₃), 1.70 (m, 4H, —CH₂CH₂CH₃), 2.99 (m,J=Hz, 4H, Pr₂NCH₂CH₂—Ar), 3.07 (m, 4H, —CH₂CH₂CH₃), 3.22 (m, 4H,Pr₂NCH₂CH₂—Ar), 3.92 (s, 2H, —CH₂C(═O)—), 3.98 (t, 2H, —OCH₂CH₂N₃), 4.48(t, 2H, —OCH₂CH₂Br), 7.14 (d, J=7.5 Hz, 1H, Ar H), 7.33 (t, J=8.0 Hz,1H, Ar H), 7.69 (d, J=8.0 Hz, 1H, Ar H).H-[(Carbamate-Ropinirole)₁₀(EOZ)₁₉₀]-COOH 20K:Azidoethyl-N-ropinirolylcarbamate hydrochloride (0.12 g, 0.293 mmol) wasdissolved in THF (15 ml). H-[(Ptyn)₁₀(Ethyl)₂₀₀]-T-PA (0.488 g, 0.024mmol) was added and the mixture was stirred to dissolve completely. CuI(0.019 g, 0.098 mmol) and triethylamine (0.014 ml, 0.098 mmol) wereadded to give a clear red solution.After stirring for 16 hours at 45° C., the mixture was quenched with 2mL of 0.1 N HCl to give a solution with pH of 3. All the volatiles wereremoved and the residue was redissolved in methanol. The resultingmixture was passed through Dowex and amberlite IR-120 column usingmethanol as an eluent. After removing methanol, the resulting aqueoussolution was extracted twice with dichloromethane (5 mL each). Theorganic solution was dried over Na₂SO₄, filtered, concentrated down to10 mL, and precipitated by adding into 70 mL of diethyl ether. Theprecipitate was filtered and dried in vacuo to giveH-[(Carbamate-Ropinirole)₁₀(Ethyl)₂₀₀]-T-PA (0.50 g, 86% yield) as, apale yellow powder. In addition to the usual polymer backbone peaks, ¹HNMR (Varian, 500 MHz, 10 mg/mL DMSO-d₆, δ) shows the polymer chaincontained an average of 6.4 units of rotigotine with major ropinirolepeaks at 0.97 (m, 6H, —CH₂CH₂CH₃), 4.62 (m, 2H, —OCH₂CH₂Br and m, 2H,—OCH₂CH₂-triazole ring), 7.19-7.39 (br m, 3H, Ar H), and 7.91 (m, 1H,triazole H).

Example 23—Synthesis of Polyethylene Glycol Dendrimer (26K)

The syntheses of the PEG dendrimer has two steps, first the building ofthe PEG dendron blocks and second the convergence of the blocks tocreate the dendrimer structure.

i. Preparation of Dendron Building Block:

Et-G1-NHBoc. L-lysine ethyl ester dihydrochloride (0.253 g, 1.025 mmol)and SCM-PEG-NHBoc 2K (4.71 g, 2.36 mmol) were dissolve indichloromethane (170 ml). After addition of TEA (0.714 ml, 5.12 mmol),the mixture was stirred overnight at room temperature. The reactionmixture was quenched with 51 mL of 0.1N HCl solution and stirred with ofNaCl (5.1 g).

Two layers were separated and the aqueous phase was extracted withdichloromethane (50 mL). The combined organic phases were dried overNa₂SO₄, filtered, concentrated using a rotary evaporator, and dried invacuo give a crude as a waxy solid. The crude was redissolved in waterand passed through an Amberlite column and then an ion-exchange columnusing both DEAE Sepharose FF and SP Sepharose FF. The resulting aqueoussolution was charged with NaCl (15% w/v) and extracted withdichloromethane. The combined organic phases were dried over anhydrousNa₂SO₄, filtered, concentrated using a rotary evaporator, and dried invacuo to provide Et-G1-NHBoc (3.4 g, 84% yield). ¹H NMR (Varian, 500MHz, 10 mg/mL CDCl₃) showed the usual backbone peak at 3.64 ppm (m, 4H,—(OCH₂CH₂)_(n)—) and other major peaks at 1.28 ppm (t, 3H, —OCH₂CH₃),1.44 ppm (s, 18H, —NHBoc), 4.01 ppm (m, 4H two protons for each PEG,—NHC(═O)CH₂—(OCH₂CH₂)_(n)—), 4.32 ppm (q, 2H, —OCH₂CH₃), 4.59 ppm (q,1H, —CH(CO₂EONH—).

CO₂H-G1-NHBoc. Et-G1-NHBoc (0.975 g, 0.247 mmol) was dissolved in water(6.2 ml) and stirred overnight with 0.1 N NaOH (5 ml, 0.5 mmol). Themixture was acidified by adding 0.5 mL of 1N HCl, charged with 1.8 g ofNaCl (15% w/v), and then stirred with 10 mL of DCM. The two layers wereseparated and the aqueous phase was extracted with 8 mL of DCM. Thecombined organic phases were dried over Na₂SO₄, filtered, concentrated,and dried in vacuo to give CO₂H-G1-NHBoc (0.928 g, 96% yield) as a paleyellow waxy powder. The completion of the hydrolysis was confirmed by ¹HNMR (Varian, 500 MHz, 10 mg/mL CDCl₃) revealed the disappearance ofester proton peaks, shown at 1.28 and 4.32 ppm (—OCH₂CH₃)Et-G1-NH2.2TFA. Et-G1-NHBoc (2.42 g, 0.613 mmol) was dissolved indichloromethane (15.33 ml) and stirred with TFA (2.36 ml, 30.7 mmol) for1 hour at room temperature. Most of the volatiles were removed using arotary evaporator to give ˜4.5 g of thick red extract. The crude wasstirred with 30 mL of diethyl ether to give a sticky powdery materialand slightly cloudy solution. After decanting the solution, the residuewas stirred with 30 mL of diethyl ether. After decanting the solution,the pale white powder (waxy) was dried over night in vacuo. The crudewas redissolved in 25 mL of dichloromethane and then washed with brine(20 mL), dried over Na₂SO₄, filtered, concentrated using a rotaryevaporator, and dried in vacuo to give Et-G1-NH₂·2TFA (2.10 g, 86%yield). The completion of the deprotection was confirmed by thedisappearance of -Boc group proton peak, shown at 1.44 ppm (s, 18H,—NHBoc).CO₂H-G1-Ethynyl. HOBT (0.209 g, 1.362 mmol) was dried by azeotrope usingacetonitrile. To the residue was added a solution of 4-pentynoic acid(0.125 g, 1.277 mmol) in dichloromethane (20 ml). DCC (0.264 g, 1.277mmol) was added and the mixture was stirred for 10 minutes to give acloudy solution. A solution of Et-G1-NH₂·2TFA (1.69 g, 0.426 mmol) withTEA (0.356 ml, 2.55 mmol) in dichloromethane (20 ml) was added. Afterstirring for 18 hours, the reaction mixture was filtered using a syringefilter and quenched with 0.1N HCl. All the organic volatiles wereremoved using a rotary evaporator and passed through an Amberlite columnand then an ion-exchange column using DEAE Sepharose FF. The resultingaqueous solution was charged with NaCl (15% w/v) and extracted withdichloromethane. The organic phase was dried over anhydrous Na₂SO₄,filtered, concentrated using a rotary evaporator, and dried in vacuo toprovide Et-G1-Ethynyl.Hydrolysis of Et-G1-Ethynyl. The ethyl ester product was dissolved inwater and pH of the solution was adjusted to 13 using 0.5 N NaOH. Afterstirring overnight, the mixture was acidified to pH of 3 and purified byan Amberlite column and an ion-exchange column using DEAE Sepharose FFto give 1.14 g (69% yield) of CO2H-G1-Ethynyl as the desired product. ¹HNMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed the usual backbone peak at3.64 ppm (m, 4H, —(OCH₂CH₂)_(n)—) and other major peaks at 2.03 (m, 2H,—CH₂CH₂CCH), 2.42 (t, 4H, —CH₂CH₂CCH), 2.53 (t, 4H, —CH₂CH₂CCH),3.98-4.16 ppm (m, 4H two protons for each PEG,—NHC(═O)CH₂—(OCH₂CH₂)_(n)—), 4.62 ppm (q, 1H, —CH(CO₂Et)NH—).ii. Construction of Dendrimer via Convergent Pathway

Et-G2-NHBoc. HOBT (0.035 g, 0.227 mmol) was dried by azeotrope usingacetonitrile (20 mL). To the residue was added a solution ofCO₂H-G1-NHBoc (0.890 g, 0.227 mmol) in dichloromethane (15 ml). DCC(0.047 g, 0.227 mmol) was added and the mixture was stirred for 3 hours.After addition of Et-G1-NH₂.2TFA (0.410 g, 0.103 mmol) and TEA (0.086ml, 0.620 mmol), the reaction mixture was stirred overnight at roomtemperature. The mixture was filtered using a syringe filter andquenched with 0.1N HCl. All the organic volatiles were removed using arotary evaporator. The resulting aqueous solution was passed through anAmberlite column and then an ion-exchange column using both DEAESepharose FF and SP Sepharose FF. The resulting aqueous solution wascharged with NaCl (15% w/v) and extracted with dichloromethane. Thecombined organic phases were dried over anhydrous Na₂SO₄, filtered,concentrated using a rotary evaporator, and dried in vacuo to provideEt-G2-NHBoc (0. 879 g, 74% yield). Ion-exchange analysis on both DEAEand SP column revealed all neutral species. ¹H NMR (Varian, 500 MHz, 10mg/mL CDCl₃) showed the usual backbone peak at 3.64 ppm (m, 4H,—(OCH₂CH₂)_(n)—) and other major peaks at 1.28 ppm (m, 3H, —OCH₂CH₃),1.44 ppm (s, 36H, —NHBoc), 3.98-4.04 ppm (m, 12H two protons for eachPEG, —NHC(═O)CH₂—(OCH₂CH₂)_(n)—), 4.19 ppm (m, 2H, —OCH₂CH₃), 4.59 ppm(q, 1H, —CH(CO₂Et)NH—).Et-G2-NH2.4HCl. Et-G2-NHBoc (0.877 g, 0.076 mmol) was stirred with 20 mLof Methanolic HCl (5 ml, 15.20 mmol) for 1 hour at room temperature. Allthe volatiles were removed by rotavap. The residue was redissolved in 30mL of dichloromethane and washed with 25 mL of brine solution. Theorganic solution was dried over Na₂SO₄, filtered, concentrated, anddried in vacuo to give Et-G2-NH₂·HCl (0.883 g, quantitative yield). ¹HNMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed the usual backbone peak at3.64 ppm (m, 4H, —(OCH₂CH₂)_(n)—) and other major peaks at 1.28 ppm (m,3H, —OCH₂CH₃), 3.94-4.04 ppm (m, 12H two protons for each PEG,—NHC(═O)CH₂—(OCH₂CH₂)_(n)—), 4.17 ppm (m, 2H, —OCH₂CH₃). The completionof the deprotection was confirmed by the disappearance of -Boc groupproton peak, shown at 1.44 ppm (s, 36H, —NHBoc).Et-G3-Ethynyl. HOBT (0.051 g, 0.332 mmol) was dried by azeotrope using30 mL of acetonitrile. To the residue was added a solution ofCO₂H-G1-Ethynyl (1.133 g, 0.292 mmol) in dichloromethane (33 ml). DCC(0.060 g, 0.292 mmol) was added and the mixture was stirred for 2 hoursat room temperature to give a cloudy solution. After addition ofEt-G2-NH2 HCl (0.75 g, 0.066 mmol) and TEA (0.074 ml, 0.532 mmol), themixture was stirred for 16 hours at room temperature. The mixture wasquenched with 6 mL of 0.1 N HCl. All the organic volatiles were removedusing a rotary evaporator and the remaining aqueous solution was dilutedwith 15 mL of water. The resulting aqueous solution was passed throughan Amberlite column and then an ion-exchange column using both DEAESepharose FF and SP Sepharose FF to remove excess acid dendron speciesand amino species due to the incompletion of the reaction. The resultingaqueous solution was charged with NaCl (15% w/v) and extracted withdichloromethane. The combined organic phases were dried over anhydrousNa₂SO₄, filtered, concentrated using a rotary evaporator, and dried invacuo to provide pale yellow solids. Further purification was performedby stirring with 30 mL of diethyl ether for 30 minutes, filtering on aglass frit, and drying to give Et-G3-Ethynyl (1.221 g, 69% yield) aspale yellow crystalline. Ion-exchange analysis on both DEAE and SPcolumn revealed all neutral species. ¹H NMR (Varian, 500 MHz, 10 mg/mLCDCl₃) showed the usual backbone peak at 3.64 ppm (m, 4H,—(OCH₂CH₂)_(n)—) and other major peaks at 1.28 ppm (m, 3H, —OCH₂CH₃),2.03 (m, 2H, —CH₂CH₂CCH), 2.43 (t, 16H, —CH₂CH₂CCH), 2.53 (t, 16H,—CH₂CH₂CCH), 3.98-4.0,3 ppm (m, 28H two protons for each PEG,—NHC(═O)CH₂—(OCH₂CH₂)_(n)—), 4.17 ppm (m, 2H, —OCH₂CH₃), 4.40 ppm (q,6H, —CH(CO—)NH—). 4.62 ppm (q, 1H, —CH(CO₂Et)-NH—).

Example 24—PEG Et-G3-Ethynyl Dendrimer 26K Attached to Rotigotine3-azidopropionate

Rotigotine 3-azido propionate (0.192 g, 0.365 mmol) and Et-G3-Ethynyl(1.077 g, 0.041 mmol) were dissolved in THF (27.0 ml). triethylamine(0.090 ml, 0.648 mmol) and CuI (0.123 g, 0.648 mmol) were added and themixture was stirred for 40 hours at 50° C. After cooling down to roomtemperature, the mixture was stirred with 12 mL of 0.1N HCl solution.After removing THF using a rotary evaporator, the resulting aqueoussolution was diluted with 10 mL of water and passed through Amberlite(IR-120H) column (50 mL) and Dowex® M4195 column (50 mL) using 0.01% HClsolution as an eluent. The collected aqueous solution was stirred with70 mL of dichloromethane using 22 g of NaCl (15 w/v % of water amount).Two layers were separated and the aqueous phase was stirred with 70 mLdichloromethane. The combined organic phases were dried over Na₂SO₄,filtered, concentrated, precipitated by adding into diethyl ether,filtered, and dried in vacuo. The resulting waxy solid was stirred withdiethyl ether (20 mL) for 1 hour, filtered, and dried to give 0.997 g(82% yield) of the desired product, Et-G3-Rotig HCl, as pale yellowpowder. ¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed the usual PEGpeak at 3.64 ppm (m, 4H, —(OCH₂CH₂)_(n)—) and other major peaks at 1.28ppm (m, 3H, —OCH₂CH₃), 3.97-4.03 ppm (m, 28H two protons for each PEG,—NHC(═O)CH₂—(OCH₂CH₂)_(n)—), 4.17 ppm (m, 2H, —OCH₂CH₃), 4.41 ppm (q,6H, —CH(CO)NH—), and 4.62 ppm (q, 1H, —CH(CO₂Et)NH—). Rotigotinyl peaksrevealed at 1.04 ppm (t, 3H, —CH₂CH₂CH₃), 4.73 ppm (m, 2H,triazole-CH₂CH₂C(═O)ORotig), 6.89-7.20 ppm (m, 6H, aromatic andthiophenyl H), 7.70 (br s, 1H, triazole H). Number of rotigotinemolecules on the dendrimer was determined as 5.6 by both ¹H NMR andreverse phase HPLC analysis. ‘Click’ reaction was monitored by thedisappearance of the termini peaks showed at 2.03 (m, 2H, —CH₂CH₂CCH)and 2.43 (t, 16H, —CH₂CH₂CCH), and by the appearance of triazole protonpeak at 7.70 ppm.

Example 25—Synthesis of mPEG-co-polyamido G2 Ethynyl Dendrimer (20K)

Fmoc-G2-ester: A 25 mL of round bottom flask was charged with 1-HOBThydrate (0.342 g, 2.24 mmol), dried by azeotrope using 15 mL ofacetonitrile. After adding DMF (8 ml), Fmoc-G1-acid (0.3 g, 0.639 mmol)and DCC (0.461 g, 2.24 mmol) were added. After stirring for 1 h 30minutes, the mixture became cloudy and amino-G1-ester (0.929 g, 2.24mmol) was added. The resulting pale yellow precipitated solution wasallowed to stir for 16 hours at room temperature. The mixture wasfiltered and the filtrate was concentrated in vacuo. The residue wasdissolved in dichloromethane (20 mL) and washed with a saturated aqueoussolution of NaHCO₃ twice (10 mL each) and then with brine. The organicphase was dried over Na₂SO₄, filtered, and concentrated using a rotaryevaporator. The crude was purified by silica gel column chromatographyeluting with a solvent mixture of EtOAc/hexanes (2:3 and then 1:1) togive 0.89 g of the desired product Fmoc-G2-ester in 84% yield. ¹H NMR(Varian, 500 MHz, 10 mg/mL CDCl₃, δ): 1.41 (s, 81H, —COOC(CH₃)₃), 1.96(m, 24H, —NHC(CH₂CH₂CO—)₃), 2.20 (m, 24H, —NHC(CH₂CH₂CO—)₃), 4.20 (t,J=6.5 Hz, 1H, CHCH₂OC(═O)NH—), 4.30 (d, J=6.5 Hz, 2H, CHCH₂OC(═O)NH—),6.03 (br s, 311, —CH₂C(═O)NH—), 6.48 (br s, 1H, —CH₂OC(═O)NH—), 7.32 (t,J=7.5 Hz, 2H, Ar H), 7.39 (t, J=7.5 Hz, 2H, Ar H), 7.66 (d, J=7.5 Hz,2H, Ar H), 7.76 (d, J=7.5 Hz, 2H, Ar H).Fmoc-G2-acid: Fmoc-G2-ester (0.89 g, 0.535 mmol) was dissolved in HCOOH(5.4 ml). After stirring for 16 hours, all the volatiles were removedusing a rotary evaporator to give a thick oily material. The residue wasstirred with diethyl ether, filtered, and dried to give a white powder(0.587 g, 95% yield). ¹H NMR (Varian, 500 MHz, 10 mg/mL CD₃OD, δ): 1.91(m, 24H, —NHC(CH₂CH₂CO—)₃), 2.28 (m, 24H, —NHC(CH₂CH₂CO—)₃), 4.23 (t,J=6.5 Hz, 1H, CHCH₂OC(═O)NH—), 4.36 (d, J=6.5 Hz, 2H, CHCH₂OC(═O)NH—),6.84 (br s, 1H, —CH₂OC(═O)NH—), 7.33 (t, J=7.0 Hz, 2H, Ar H), 7.40 (t,J=7.0 Hz, 2H, Ar H), 7.70 (d, J=7.0 Hz, 2H, Ar H), 7.80 (d, J=7.0 Hz,2H, Ar H). The completion of hydrolysis was confirmed by thedisappearance of tent-butyl group peak showing at 1.41 ppm.amino-G2-ethynyl: Propargyl amine (0.415 g, 7.54 mmol), clear yellowoil, was weighed in a 100 mL round bottom flask and then diluted withDMF (38 ml). Fmoc-G2-ester (0.436 g, 0.377 mmol) was added to give acrowded solution. TBTU (1.45 g, 4.52 mmol) was added to give a clearyellow solution. After addition of TEA (1.26 ml, 9.05 mmol), thereaction mixture was allowed to stir for 4 days at room temperature. Allthe volatiles were removed in vacuo and the residue was stirred with 40mL of dichloromethane to give a cloudy solution. The resulting mixturewas stirred with brine solution (25 mL) resulting in two layersseparation with a yellow sticky precipitate. Both organic and aqueoussolutions were decanted and the residual yellow sticky material wasdissolved in methanol. The recovered solution in methanol wasconcentrated and dried in vacuo to give 0.358 g of the desiredamino-G2-ethynyl as pale yellow powder in 75% yield. ¹H NMR (Varian, 500MHz, 10 mg/mL CD₃OD, δ): 1.70 (br t, 6H, NH₂C(CH₂CH₂CO—)₃), 2.00 (m,18H, —NHC(CH₂CH₂CO—)′₃), 2.21 (m, 24H, —C(CH₂CH₂CO—)₃), 2.60 (s, 9H,—NHCH₂CCH), 3.96 (d, J=2.0 Hz, 2H, —NHCH₂CCH). The completion of Fmocgroup deprotection was confirmed by the disappearance of Fmoc grouppeaks.mPEG-co-polyamido-G2-ethynyl. mPEG-SVA 20K (0.429 g, 0.021 mmol) andamino-G2-ethynyl (0.0404 g, 0.032 mmol) were dissolved in 6 mL of 1:1DMF/dichloromethane. After addition of TEA (0.012 ml, 0.085 mmol), themixture was stirred for 18 hours at room temperature. All the volatileswere removed in vacuo at 40° C. and the residue was redissolved in 4 mLof DCM to give a milky solution. Upon the addition of IPA (12 mL), thesolution became clear. Dichloromethane was removed using a rotavap togive a solution with white precipitates. After stirring for 10 minute atroom temperature, the white precipitates were filtered, washed with IPA,and dried in vacuo to give 0.432 g (95% yield) ofmPEG-polyamido-G2-ethynyl, block copolymer of PEG and polyamidodendrimer. ¹H NMR (Varian, 500 MHz, 10 mg/mL CD₃OD, δ): 1.70 (m, 2H,mPEG-CH₂CH₂CH₂CH₂C(═O)—), 1.82 (m, 2H, mPEG-CH₂CH₂CH₂CH₂C(═O)—), 1.94(br t, 6H, —NHC(CH₂CH₂CO—)₃), 2.01 (m, 18H, —NHC(CH₂CH₂CO—)₃), 2.21 (m,18H, —C(CH₂CH₂CO—)₃), 2.38 (m, 6H, —C(CH₂CH₂CO—)₃), 2.62 (s, 9H,—NHCH₂CCH), 3.37 (s, 3H, CH₃O—), 3.64 (m, PEG backbone,CH₃O(CH₂CH₂O)_(n)CH₂—), 3.97 (br s, 2H, —NHCH₂CCH).

Example 26—PEG-Polyamido Dendrimer Attached to Rotigotine3-azidopropionate

Rotigotine 3-azidopropionate·HCl (0.085 g, 0.189 mmol) was dissolved inTHF (12 ml). mPEG-polyamidoG2-ethynyl derndrimer (0.426 g, 0.020 mmol)was added and the mixture was stirred to dissolve completely. CuI (0.014g, 0.072 mmol) and triethylamine (0.039 ml, 0.278 mmol) were added andthe mixture was stirred for 16 hours at 45° C. After cooling down toroom temperature, the mixture was quenched with 10 mL of 0.1N HClsolution. All the organic volatiles were removed using a rotaryevaporator. The resulting aqueous solution was diluted with 10 mL ofmethanol and then passed through Dowex® M4195 column (15 mL) followed bymethanol washing. After removing methanol using a rotary evaporator, theresulting aqueous solution was stirred with dichloromethane (20 mL each)twice using 1 g of NaCl. The combined organic phases were dried overNa₂SO₄, filtered, concentrated, and precipitated by adding into diethylether. The precipitated solution was filtered, and dried to give 0.47g(quantitative yield) of the desired product, mPEG-polyamidoG2-Pr-Rotig,as a pale yellow crystalline material. Besides the copolymer backbonepeaks, ¹H NMR (Varian, 500 MHz, 10 mg/mL CD3OD, δ) showed majorrotigotinyl peaks, due to the completion of ‘click’ reactions, at 1.05ppm (d, 27H, Rotigotinyl —CH₂CH₂CH₃), 4.40 ppm (m, 18H,—NtriazoleCH₂CH₂C(═O)O-Rotig), 4.70 ppm (m, 18H,—C(═O)NHCH₂—Ctriazole-), and 7.94 ppm (s, 9H, triazole H).

Example 27—Synthesis of Oxidized Polydextran (20K)

Polyal (Oxidized Dextran) Synthesis: 5.58 g of sodium periodate (26mmole) was dissolved in 30 mL of DI-H₂O in a 100 mL one-neckround-bottom flask. The flask was covered with aluminum foil. In a 20 mLvial, 2.0 g of dextran (0.13 mmole, M_(n): 15,340 g/mole, M_(p): 22,630g/mole, PD: 2.11) was dissolved in 15 mL of DI-H₂O and this solution wasslowly added into the round-bottom flask. The vial was rinsed with 15 mLof DI-H₂O and the rinse solution was also added into the round-bottomflask. The clear colorless solution was stirred at room temperature for24 h. At the end of this time, the aqueous solution was transferred intotwo Slide-A-Lyzer 2K dialysis cassettes and dialysis was conducted inwater overnight. This aqueous solution (˜60 mL) was used in the nextstep.Polyalcohol Synthesis from Polyal: 1.134 g of sodium borohydride (30mmole) was dissolved in 10 mL of DI-H₂O in a 100 mL one-neckround-bottom flask. The aqueous solution from the previous step(BD-29-8) was then added slowly into the round-bottom flask. Thesolution was stirred for 18 h. The pH of the solution was adjusted to 6using 3M HCl and the solution was again dialyzed using three 10K MWCOdialysis cassettes and for two days. The aqueous solution wasconcentrated down to 5 mL and then lyophilized for two days to give 1.56g of the polyalcohol in 94% yield.¹H NMR (DMSO-d6, δ, ppm, TMS): 3.35 (2H, —OCH₂CH(CH₂OH)O—), 3.48 (2H,—OCH(CH₂OH)O—), 3.58-3.70 (2H, —OCH₂CH(CH₂OH)O—), 3.64 (1H,—OCH₂CH(CH₂OH)O—), 4.62 (2H, —OCH₂CH(CH₂OH)OCH(CH₂OH)O—), 4.70 (1H,—OCH(CH₂OH)O—). ¹³C NMR (DMSO-d6, δ, ppm, TMS): 64.56(—OCH₂CH(CH₂OH)O—), 65.10 (—OCH(CH₂OH)O—), 68.96 (—OCH₂CH(CH₂OH)O—),79.88 (—OCH₂CH(CH₂OH)O—), 105.86 (—OCH(CH₂OH)O—). GFC: M_(n): 11,100g/mole, M_(p): 19,270 g/mole, PD: 2.41

Polyalcohol Propargyl bromide Reaction: 840.0 mg of polyalcohol (5×10⁻⁵mole, M_(n): 11,100 g/mole, M_(p): 19,270, PD: 2.4) was dissolved in 10mL of dimethylformamide in a 25 mL round-bottom flask. 5 mL of toluenewas then added into the round-bottom flask. Toluene was rotovapped downat 50° C. at 40 mbar using a rotary evaporator. 407.5 mg of cesiumcarbonate (1.25×10⁻³ mole) was then added into the round-bottom flask.The mixture was stirred for 3 h under Argon at 60° C. 234.0 mg ofpropargyl bromide solution (80% solution in toluene, 187.5 mg ofpropargyl bromide, 1.25×10⁻³ mole) was added into the round-bottomflask. The cloudy solution was stirred at 60° C. for 34 h under Argon.At the end of this time, the yellow cloudy solution was cooled down toroom temperature, filtered through a 30 mL frit, and the filtrate wasconcentrated down to dryness. The polymer was redissolved in 15 mL ofDI-H₂O and washed with dichloromethane twice (2×45 mL). Thedichloromethane phase was washed with 15 mL of DI-H₂O. Aqueous phaseswere separated, combined and rotovapped down to remove any residualdichloromethane. The aqueous solution was then dialyzed using a 2K MWCOdialysis cassette overnight. The water was removed and the polymer wasdried under high vacuum to give 730.0 mg of the final product.¹H NMR (DMSO-d6, δ, ppm, TMS): 3.35 (2H, —OCH₂CH(CH₂OH)O—), 3.48 (2H,—OCH(CH₂OH)O—), 3.58-3.70 (2H, —OCH₂CH(CH₂OH)O—), 3.64 (1H,—OCH₂CH(CH₂OH)O—), 4.18 (4H, —OCH₂CH(CH₂OCH₂C≡CH)OCH(CH₂OCH₂CCH)O—),4.62 (2H, —OCH₂CH(CH₂OH)OCH(CH₂OH)O—), 4.70 (1H, —OCH(CH₂OH)O—). FromNMR data, the average value of ‘n’ is 78 and of ‘m’ is 5.

Example 28—Oxidized Polydextran (20K) Attachment to 3-azidopropylRotigotine

Three hundred and forty two milligrams (342.0 mg) of 3-azidopropionylrotigotine.TFA (6.5×10⁻⁴ mole) was weighed in a 100 mL round-bottomflask and 835.0 mg of oxidized dextran with acetylene pendents (6.5×10′mole; average ‘n’ value of 89, ‘m’ value of 6) and was added into theflask. Eighty milliliters (80 mL) of dimethylformamide was then addedinto the flask to completely dissolve the polymer. 64.5 mg of coppersulfate (2.6×10⁴ mole) and 103.0 mg of sodium ascorbate (5.2×10⁻⁴ mole)were then added into the round-bottom flask. The round-bottom flask wasclosed with a rubber septum and the solution was stirred at 40° C. underArgon overnight. More copper sulfate (258.0 mg, 1.04×10⁻³ mole) andsodium ascorbate (412.0 mg, 2.08×10⁻³ mole) were added into the RBF andthe solution was stirred overnight at 40° C. More copper sulfate (322.5mg, 1.3×10⁻³ mole) and sodium ascorbate (515.0 mg, 2.6×10⁻³ mole) wereadded into the RBF and the solution was stirred overnight at 40° C. Atthe end of this time; the solution was cooled down to room temperature,filtered through a coarse fit, and rotovapped down to dryness. Theresidue was redissolved in 60 mL of DMF, filtered, concentrated down to10 mL and precipitated into diethyl ether (200 mL). The solvents weredecanted and the polymer was dried under high vacuum overnight to give362.0 mg of the final product. ¹H NMR (DMSO-d6, δ, ppm, TMS): 0.86 (3H,—NCH₂CH₂CH₃); 1.4-3.6 (total of 17H, aliphatic CH and CH₂ peaks ofrotigotine); 3.36 (2H, —OCH₂CH(CH₂OH)O—), 3.47 (2H, —OCH(CH₂OH)O—),3.57-3.70 (2H, —OCH₂CH(CH₂OH)O—), 3.64 (1H, —OCH₂CH(CH₂OH)O, 4.62 (2H,—OCH₂CH(CH₂OH)OCH(CH₂OH)O—), 4.70 (1H, —OCH(CH₂OH)O—); 6.80-7.29 (6H,—CH peaks of 1,2,3,4-tetrahydronaphtalene and —CH peaks of 2-thiophene);8.14 (1H, —CH peak of triazole).

Example 29—Hydrolysis of Active Drug Molecules (Rotigotine, Etoposide,Irinotecan, Tiagabine) from their Polymer Conjugated Forms

The cleavage of rotigotine, etoposide, irinotecan and tiagabine from thedifferent types of linkers attached to the backbones of polyoxazoline,polyethylene glycol, modified dextran and PEG dendrimer polymers wasexamined in rat plasma. Four milliliters of rat plasma was placed in atest tube, and then spiked with approximately 16 mg of each polymer drugconjugate dissolved in 400 μL of a 5% dextrose solution. The test tubeswere placed in a 37° C. water bath and allowed to incubate forapproximately 48-72 hours. At regular time intervals, a 100 μL aliquotof plasma was taken and placed in a 1.5 mL centrifuge tube, neutralizedwith 5 μL of dilute acid solution (3M HCl), and treated withapproximately 500 μL of acetonitrile to precipitate the plasma proteinsand dissolve the released drug. The tube was centrifuged at 14,000 rpmfor 5 minutes. The supernatant was removed, diluted in 0.1% TFA inwater, filtered, placed in a HPLC vial, and assayed by reverse phasechromatography using a Zorbax C8 300SB, 5μ, 4.6×150 mm column fixed toan Agilent 1100/1200 chromatogarphy system fitted with a variable UVdetector set at a wavelength to accommodate for the λmax of each drug.The mobile phase was 0.1% TFA in water (A) and 0.1% TFA in acetonitrile(B) eluting a rate of 1 mL/min. A standard curve was created by spikinga known concentration of drug in plasma and extracting and assaying thefree drug as described above. The amount of drug in each aliquot wascalculated from the standard curve above and a plot of the concentrationof drug released versus time was generated. The half-life of eachpolymer drug conjugate was calculated and reported in Tables 1-3.

TABLE 1 Effect of linker and polymer on rate of release of rotigotinefrom rotigotine esters (polymer-triazine-alkyl-CO—O-Rotigotine) inplasma, pH 7.4, 37° C. % Drug Polymer* Alkyl Linker Loading Half-LifePOZ —CH₂— 14.2 2.4 ± 0.28 hours (for n = 2) POZ —CH₂(CH₃)— 9.6 7.1 hoursPOZ —CH₂CH₂— 13.0 11.9 ± 4.2 hours (for n = 6) POZ —CH₂CH₂CH₂— 12.4 5.0hours PEG —CH₂CH₂— 5.2 8 minutes PEG —CH₂CH₂— 5.4 11 minutes DendrimerModified —CH₂CH₂— 2.3 <2 minutes Dextran *POZ is MW 20,000, acidterminus, 10 triazine pendents. PEG is four arm, MW 20,000, fourtriazine terminae. See text for structures.

TABLE 2 Effect of drug on rate of release of drug from POZ-triazine-CH₂—CO—O-Drug in plasma, pH 7.4, 37° C. % Drug Drug LoadingHalf-Life (hours) Etoposide 18.2 3.9 Irinotecan 16.5 6.5 Rotigotine 13.72.4 Tiagabine 14.5 80.6 POZ is MW 20,000, acid terminus, 10 triazinependents.

TABLE 3 Effect of molecular weight and number of pendents on cleavagerate of POZ-triazine-CH₂—CO—O-Irinotecan in 50 mM sodium phosphate, pH7.4, 37° C. Pendents MW Half-Life (hours) 10 20K 8.6 20 20K 8.3 20 30K9.4 20 40K 7.6

The results shown in Table 1 demonstrate that the length of the linkerinfluences the rate of release of the agent, in this case rotigotine,from the polyoxazoline conjugate. The results show that as the length orsize of the azidoalkyl acid linker increases, the rate of release ofrotigotine from the polyoxazoline conjugate decreases. Table 2 showsthat the nature of the agent also impacts the rate of release of theagent from the polymer. Table 3 shows that the molecular weight and thenumber of pendants groups do not significantly affect the rate ofrelease when of irinotecan from polyoxazoline. Taken together, theresults show that the release of an agent from a polyoxazoline conjugatecan be tuned to release desired amounts of the agent over time.

TABLE 4 Effect of linker and polymer on rate of release of tiagabinefrom tiagabine esters (Polymer-triazine-linker-O—CO-tiagabine) inplasma, pH 7.4, 37° C. % Drug Half-Life Polymer* Linker Loading (days)POZ —CH₂—CH₂— 14.7 4.6 POZ —CH₂—CH₂CH₂— 13.8 3.8 POZ —(CH₂CH₂O)₃— 14.22.8 POZ —CH₂—CH₂—CO—NH—(C₆H₄)— 11.3 6.9 PEG —CH₂—CH₂CH₂— 10.4 0.5 *POZis MW 20,000, acid terminus, 10 triazine pendents. PEG is four arm, MW10,000, four triazine terminae. See text for structures.

The release of tiagabine from a 20K polyoxazoline and 10K polyethyleneglycol using three different types of linkers was also determined. Thetypes of linkers tested were the alkyl linker, a polyethylene glycollinker and an aromatic amide linker for the polyoxazoline conjugates andthe alkyl linker for the polyethylene glycol conjugate. Table 4summarizes the drug loading % and approximate release half-lives (days).The results show that the more hydrophilic PEG polymer shows a fasterdrug release profile consistent with the results shown in Table 1.

While not being bound by any particular theory, it is hypothesized thatthe surprisingly slow hydrolysis rate of the compounds illustrated inTables 1, 2 and 4 may result from the folding of the polymer to providea water-poor environment for the bound drug and its associatedreleasable linker. In contrast, the relatively rapid hydrolysis of thePOZ conjugate containing a ethylene oxide units as a linker may beexplained by the assumption that the ethylene oxide units of theoligo(ethylene oxide) linker bring water into the neighborhood of thecleavable moiety. It is known from independent studies that there are2-4 water molecules associated with each ethylene oxide unit ofpoly(ethylene oxide) (also known as PEG). In other words, the bound drugand its associated releasable linker reside in a water-rich environmentrather than a water-poor environment as is the case in the otherconjugates studied.An alternative explanation is that one of the oxygen atoms of theethylene oxide units could act to give a “neighboring groupparticipation” effect. Neighboring group participation is a well-knowntheory to explain the ability of neighboring atoms to act as internalnucleophiles and speed up the cleavage of groups such as esters.

Example 30—Comparative Viscosity of Different Polymer Conjugates

The viscosity of each polymer drug conjugated sample was measured on aBrookfield LVDV-II Cone and Plate viscometer fitted with a temperaturecontrolled jacketed plate. The polymer sample (0.5 mL of a 10, 20, 30and 40% w/w solution in water) was placed on the center of the plate,which was attached to the main drive of the instrument. The cone(CPE-40) was rotated at different rates (rpm) and the viscosity (mPas)was recorded each time at 25° C. The table below shows a comparison ofviscosity readings for each sample tested. The results show that POZconjugates of the present disclosure have low viscosity that allow forease of administration through a narrow bore needle.

TABLE 5 Viscosity of Polymer Conjugates of rotigotine, measured at 25°C. Syringeability through Viscosity 28G needle (150 g PolymerConcentration Drug Content (mPas) pressure) POZ - Rotigotine 30% 40mg/mL 64.8 Yes 20K PEG - Rotigotine 52% 50 mg/mL 120.7 Yes 10K PEG -Rotigotine 50% 25 mg/mL 217.5 No 20K PEG Dendrimer - 50% 27 mg/mL 142.3Yes Rotigotine 20K Modified Dextran - 50% 23 mg/mL 160.0 Yes Rotigotine20K POZ - tiagabine 20K 40% 55.2 mg/mL   200.6 Yes PEG - tiagabine 10K40% 41.6 mg/mL   73.0 Yes

Example 31—Pharmacokinetics of Rotigotine in Rat After Intravenous andSubcutaneous Administration of H-[(Acetyl-Rotigotine)₁₀(EOZ)₁₉₀]-COOH20K and H-[(Propionyl-Rotigotine)₁₀(EOZ)₁₉₀]-COOH 20K

In order to study the pharmacokinetics of the POZ conjugates describedherein, in vivo studies were conducted with male Sprague-Dawley rats.Twenty-seven male cannulated Sprague-Dawley rats (300-350 g) weredivided into nine groups of 3 animals per group. Groups I-II received asingle subcutaneous (SC) dose (right flank) of POZ acetyl rotigotine (asdescribed in Example 6) at equivalent doses of 1.6 and 6.4 mg/kg. GroupsIII-IV received a single subcutaneous (SC) dose (right flank) of POZpropyl rotigotine (as described in Example 7) at equivalent doses of 1.6and 6.4 mg/kg. Group V received a single subcutaneous (SC) dose (rightflank) of rotigotine hydrochloride at an equivalent dose of 0.5 mg/kg.Groups VI-VII received a single intravenous (IV) dose (lateral tailvein) of POZ acetyl rotigotine (as described in Example 6) at equivalentdoses of 0.5 and 2.0 mg/kg. Groups VIII-IX received a single intravenous(IV) dose (lateral tail vein) of POZ propyl rotigotine (as described inExample 7) at equivalent doses of 0.5 and 2.0 mg/kg. The test articleswere dissolved in 5% dextrose injection and filtered prior to eachinjection. Serial blood samples were obtained from each intravenouslydosed animal through the cannulated catheter, at time intervals of endof injection, 12, 24, 48, 96 and 168 hours. The time intervals for thesubcutaneously dosed animals were 6, 12, 24, 48, 96 and 168 hours. Theblood was processed to collect the plasma which was stored at −70° C.before analysis. The plasma samples were extracted with acetonitrileusing d3-rotigotine as an internal standard and the analytes in theextract were assayed by chromatographic analysis on LC/MS-MS systemusing a C-18 reverse phase column with 0.9 um silica coreshell(Accucore™, Thermo Scientific, 30×2.1 mm ID and 2.6 micron particlesize). The mobile phase was ammonium formate 10 mM pH3.0 (solvent A);and 90% acetonitrile, 10% methanol, and 0.1% formic acid (solvent B),eluting at 0.6 mL/min.

The plasma concentration of rotigotine (ng/mL) after intravenous andsubcutaneous injection is shown in FIGS. 2 and 3, respectively. Theseresults suggest that POZ conjugates of rotigotine, whether dosedintravenously or subcutaneously, will reduce the clearance rate ofrotigotine from the blood when compared to the parent molecule alone.The terminal plasma half-life (VA) for rotigotine, POZ acetyl rotigotineand POZ propyl rotigotine was 2.8, 16 and 60 h, respectively. However,there is a striking difference in the PK profiles when thePOZ-conjugates POZ acetyl rotigotine and POZ propyl rotigotine whencompared IV vs SC. POZ-conjugates delivered IV are generally cleared ina bi-phasic pattern with little difference between POZ acetyl rotigotineand POZ propyl rotigotine. However, when the two are compared followingSC administration there is a marked difference. POZ acetyl rotigotinehas essentially the same PK profile when delivered either SC or IV. POZpropyl rotigotine has a markedly prolonged PK profile that is near “zeroorder” kinetics. The size and length of the linker plays a role in therelease of the agent, in this case rotigotine, and the levels measuredin rat plasma from day 1 to day 7 are higher for the propyl linker thanthe acetyl linker. The initial plasma concentrations of rotigotineduring the first 12 hours are lower for POZ propyl rotigotine whencompared to the POZ acetyl rotigotine compound. At 12 hours, the C_(max)values of plasma rotigotine were 6 ng/mL for POZ propyl rotigotineversus for 48 ng/mL for the POZ acetyl rotigotine when dosed SC at thedose of 1.6 mg/kg. This suggests that controlled delivery of an agentcan be “tuned” to release the agent with a desired release profilewithout an initial burst effect based on the nature of the releasablelinker, the size of the POZ polymer, the route of administration (e.g.subcutaneous) or a combination of the foregoing.

Example 32—Pharmacokinetics of Rotigotine in Monkey After SubcutaneousAdministration of H-[(α-Methyl-Acetyl-Rotigotine)₁₀(EOZ)₁₉₀]-COOH 20Kand H-[(Propionyl-Rotigotine)₁₀(EOZ)₁₉₀]-COOH 20K

The pharmacokinetics of the POZ conjugates of rotigotine was measured innormal, treatment-naïve female macaques. Animals were randomly assignedinto four treatment groups, each N=3. Animals received one subcutaneousdose of either POZ alpha methyl acetyl rotigotine (as described inExample 8) or POZ propyl rotigotine (as described in Example 7) at dosesof either 1.5 mg/kg or 4.5 mg/kg (based on rotigotine equivalents). Thetest articles were dissolved in 5% dextrose injection and filtered priorto each injection. Serial venous blood samples were obtained from eachanimal prior to administration of experimental agents on Day 1 andsubsequently at 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 24 h, 48 h, 96h, 192 h, 240 h and 336 h. The blood was processed to collect the plasmawhich was stored at −70° C. before analysis. These plasma samples wereprocessed and assayed by chromatographic analysis on LC/MS-MS system asdescribed in Example 31.

The plasma concentration of rotigotine (ng/mL) after subcutaneousinjection is shown in FIG. 4. These results show that POZ conjugates ofrotigotine will reduce the clearance rate of rotigotine from the blood.The average terminal plasma half-life (t½) of rotigotine from POZ alphamethyl acetyl rotigotine and POZ propionyl rotigotine was 9 and 60 h,respectively. Once again, the POZ propyl rotigotine has a markedlyprolonged PK profile that is near “zero order” kinetics. The initialplasma concentrations of rotigotine during the first 12 hours are lowerfor POZ propyl rotigotine when compared to the POZ alpha methyl acetylrotigotine compound. From 4 to 192 hours, the average C_(ss) value ofplasma rotigotine was between 1 and 6 ng/mL for POZ propyl rotigotine atthe 1.5 mg/kg dose.

Example 33—Efficacy of H-[(Acetyl-Rotigotine)₁₀(EOZ)₁₉₀]-COOH 20K andH-[(Propionyl-Rotigotine)₁₀(EOZ)₁₉₀]-COOH 20K in the 6-OHDA Rat ModelFollowing Subcutaneous Administration

In order to study the efficacy of the POZ conjugates described herein,in vivo studies were conducted with female Sprague-Dawley rats. FemaleSprague-Dawley rats (275-350 g) were used in the study. Each animalunderwent stereotaxic surgery and received a unilateral lesion of theright nigrostriatal pathway via injection of 12.5 μg of6-hydroxydopamine (6-OHDA) into a single site in the medial forebrainbundle. Rats were monitored over two weeks and underwent behavioralassessment (on day -7) via the cylinder test. Animals lacking overtbehavioral asymmetry (>85% ipsilateral forelimb use) were excluded fromthe study. The rats were them randomly assigned to one of six treatmentgroups (each N=8). The groups were as follows: vehicle control (GroupA); rotigotine hydrochloride 0.5 mg/kg (Group B); rotigotinehydrochloride 3 mg/kg (Group C); H-[(Acetyl-Rotigotine)₁₀(EOZ)₁₉₀]-COOH20K (as described in Example 6) 1.6 mg/kg (Group D);H-[(Propionyl-Rotigotine)₁₀(EOZ)₁₉₀]-COOH 20K (as described in Example7) 1.6 mg/kg (Group E); and H-[(Propionyl-Rotigotine)₁₀(EOZ)₁₉₀]-COOH20K (as described in Example 7) 6.4 mg/kg (Group F). The rats received asingle subcutaneous dose (2 mL/kg) of vehicle (5% dextrose) or testcompound dissolved in 5% dextrose.

The results are presented in Table 6. All treatments show positiverotational behaviors (contraversive turns) on day 1 of dosing. Only POZpropyl rotigotine shows activity on day 5, with marked and continuouscontraversive rotations at the high dose of 6.4 mg/kg. This favorableresponse is due to the high and sustained rotigotine drug levels inblood on day 5, which was observed in the pharmacokinetic study (Example32).

Each group of animals (A-F as described above) were independentlyassessed rat for rotational behavior and forelimb symmetry on day 1, day2, day 5 and day 9. In the rotational test, the animals were placed inan automated rotometer apparatus (MedAssociates, USA) and the net numberof rotations contraversive to the lesion were recorded over a period of6 hours on each day. In the forelimb symmetry test, the rats are placedin a clear glass cylinder without top (15 cm diameter×45 cm tall). Thenumber of times each paw touches the side of the cylinder during anindividual rear is recorded over a 10 minute observation on each day.The first limb in any rear to touch the wall is scored a single point.If both limbs contact within 0.4 s of each other, then this is scored asa ‘both’. All subsequent exploratory movements about the wall using thatlimb are scored independently until the other limb contacts the wallwith weight support. Alternating stepping motions involving both pawsone after the other receive a single score for both. The net number ofcontralateral touches are calculated and considered a favorableresponse.

The results are presented in Table 7. All treatments show positiveipsiversive forelimb use on day 1 of dosing. Only POZ propyl rotigotineshows activity on day 5, with marked and continuous ipsiversive forelimbuse at the both doses of 1.6 and 6.4 mg/kg. This favorable response isdue to the high and sustained rotigotine drug levels in blood on day 5,which was observed in the pharmacokinetic study (Example 32).

The following table 6 summarizes the results of the rotational test:

TABLE 6 Net number of contraversive turns/6 h period Dose (Average ±SEM; n = 8) Compound (mg/kg) Day 1 Day 5 Vehicle 0 −56 ± 20  −25 ± 11Rotigotine 0.5 983 ± 405 −49 ± 9  Rotigotine 3.0 1570 ± 312* −39 ± 15POZ Acetyl Rotigotine 20K 1.6 872 ± 232 −14 ± 14 POZ PropionylRotigotine 20K 1.6 1408 ± 286*  68 ± 60 POZ Propionyl Rotigotine 20K 6.41272 ± 405*  5142 ± 777** */**represents P < 0.01 or P < 0.001 cf.vehicle (1-way ANOVA with Dunnett's post-hoc test).

The following table 7 summarizes the results of the forelimb asymmetrytest:

TABLE 7 Net ipsiversive forelimb use as a percentage of total forelimbuse Dose (Average ± SEM; n = 8) Compound (mg/kg) Day 2 Day 5 Vehicle 088 ± 7% 85 ± 6% Rotigotine 0.5 60 ± 13% 94 ± 6% Rotigotine 3.0  9 ± 13%*85 ± 8% POZ Acetyl Rotigotine 20K 1.6 50 ± 13% 85 ± 10% POZ PropylRotigotine 20K 1.6  0 ± 14%** 31 ± 13%* POZ Propyl Rotigotine 20K 6.4 −2± 26%** −6 ± 16%** */**represents P < 0.01 or P < 0.001 cf. vehicle(1-way ANOVA with Dunnett's post-hoc test).

What is claimed:
 1. A method for treating a disease or condition relatedto dopamine insufficiency in the peripheral or central nervous system ina subject, the method comprising the step of administering to thesubject a poly(oxazoline) polymer conjugate comprising a water solublepoly(oxazoline) polymer and an agent, wherein a release profile of theagent is selectable based on the selection of the poly(oxazoline)polymer conjugate, the poly(oxazoline) polymer conjugate having thestructure:

wherein L is

R₃ is a forms a linkage with the poly(oxazoline) polymer; R₄ is—CH₂—C(O)—O—, —CH₂(CH₃)—C(O)—O—, —CH₂—CH₂—C(O)—O—,—CH₂—CH₂—CH₂—C(O)—O——CH₂—O—C(O)—, —CH₂(CH₃)—O—C(O)—, —CH₂—CH₂—O—C(O)— or—CH₂—CH₂—CH₂—O—C(O)—; R is an initiating group; R₁ is a non-reactivegroup; A is the agent; a is ran which indicates a random copolymer orblock which indicates a block copolymer; o is from 1-50; m is from1-1000; and T is a terminating group, wherein the release profile isdependent on the selection of R₃.
 2. The method of claim 1, wherein R₄is —C(O)—(CH₂)₃—.
 3. The method of claim 1 wherein L has the structure


4. The method of claim 1, wherein T is —Z—B-Q wherein Z is S, O, or N; Bis an optional linking group; and Q is a terminal portion of aterminating nucleophile.
 5. The method of claim 1, wherein R ishydrogen, alkyl or substituted alkyl.
 6. The method of claim 1, whereinthe disease or condition is Parkinson's disease or restless legsyndrome.
 7. The method of claim 1, wherein the agent is a dopamineagonist, an adenosine A_(2A) antagonist, an anticholinergic, a monamineoxidase-B inhibitor or a catechol-O-methyl transferase inhibitor.
 8. Themethod of claim 1, wherein the agent is rotigotine.
 9. The method ofclaim 1, wherein the polymer conjugate is administered alone or as apart of a pharmaceutical composition.
 10. The method of claim 1, whereinthe polymer conjugate is administered in a therapeutically effectiveamount.
 11. The method of claim 1, wherein the polymer conjugate isadministered by subcutaneous administration.
 12. A method for treating adisease or condition characterized by excessive GABA re-uptake or GABAre-uptake in a subject, the method comprising the step of administeringto the subject a poly(oxazoline) polymer conjugate comprising a watersoluble poly(oxazoline) polymer and an agent, wherein a release profileof the agent is selectable based on the selection of the poly(oxazoline)polymer conjugate, the poly(oxazoline) polymer conjugate having thestructure:

wherein L is

R₃ is a forms a linkage with the poly(oxazoline) polymer; R₄ is—CH₂—C(O)—O—, —CH₂(CH₃)—C(O)—O—, —CH₂—CH₂—C(O)—O—, —CH₂—CH₂—CH₂—C(O)—,—CH₂—O—C(O)—, —CH₂(CH₃)—O—C(O)—, —CH₂—CH₂—O—C(O)— or—CH₂—CH₂—CH₂—O—C(O)—; R is an initiating group; R₁ is a non-reactivegroup; A is the agent; a is ran which indicates a random copolymer orblock which indicates a block copolymer; o is from 1-50; m is from1-1000; and T is a terminating group, wherein the release profile isdependent on the selection of R₄.
 13. The method of claim 12, wherein R₃is —C(O)—(CH₂)₃—.
 14. The method of claim 12, wherein L has thestructure


15. The method of claim 12, wherein T is —Z—B-Q wherein Z is S, O, or N;B is an optional linking group; and Q is a terminal portion of aterminating nucleophile.
 16. The method of claim 12, wherein R ishydrogen, alkyl or substituted alkyl.
 17. The method of claim 12,wherein the disease or condition is an anxiety disorder, a socialanxiety disorder, a panic disorder, neuropathic pain, fibromyalgia,chronic pain, a muscle tremor, a muscle spasm, a seizure, a convulsionor epilepsy.
 18. The method of claim 12, wherein the agent is a GABAre-uptake inhibitor.
 19. The method of claim 12, wherein the agent isnipecotic acid or tiagabine.
 20. The method of claim 12, wherein thepolymer conjugate is administered alone or as a part of a pharmaceuticalcomposition.
 21. The method of claim 12, wherein the polymer conjugateis administered in a therapeutically effective amount.
 22. The method ofclaim 12, wherein the polymer conjugate is administered by subcutaneousadministration.